Therapeutic compounds

ABSTRACT

The present invention is directed to RNA interference (RNAi) molecules targeted against a Huntington&#39;s disease nucleic acid sequence, and methods of using these RNAi molecules to treat Huntington&#39;s disease.

CLAIM OF PRIORITY

This application is a continuation of U.S. patent application Ser. No.13/985,023, filed on Aug. 12, 2013, which is a U.S. National PhaseApplication of International Patent Application No. PCT/US2012/024904,filed on Feb. 13, 2012, which claims priority to U.S. ProvisionalApplication No. 61/442,218, filed on Feb. 12, 2011 and U.S. ProvisionalApplication No. 61/522,632, filed on Aug. 11, 2011. These applicationsare incorporated by reference herein.

GOVERNMENT SUPPORT

The invention was made with Government support under NS-50210,NS-068099, and DK-54759 awarded by the National Institutes of Health.The government has certain rights in the invention.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted in ASCII format via EFS-Web and is hereby incorporated byreference in its entirety. Said ASCII copy, is named 17023.113US1_SL.txtand is 51,200 bytes in size.

BACKGROUND OF THE INVENTION

RNAi directs sequence-specific gene silencing by double-stranded RNA(dsRNA) which is processed into functional small inhibitory RNAs(˜21nt). In nature, RNAi for regulation of gene expression occursprimarily via small RNAs known as microRNAs (miRNAs). Mature microRNAs(˜19-25 nts) are processed from larger primary miRNA transcripts(pri-miRNAs) which contain stem-loop regions. Via a series of processingevents catalyzed by the ribonucleases, Drosha and Dicer, the miRNAduplex region is liberated and a single strand (the antisense “guide”strand) is then incorporated into the RNA Induced Silencing Complex(RISC), thus generating a functional complex capable of base-pairingwith and silencing target transcripts. The mode of target repressionprimarily depends upon the degree of complementarity; transcriptcleavage typically requires a high-degree of base-pairing, whereastranslational repression and mRNA destabilization occurs when small RNAsbind imperfectly to target transcripts (most often in the 3′ UTR).Indeed for the latter, short stretches of complementarity—as little as 6bp—may be sufficient to cause gene silencing.

SUMMARY OF THE INVENTION

The present invention provides an isolated miRNA shuttle vector thatexpresses a therapeutic siRNA with limited off target toxicity. Incertain embodiments, embedding an siRNA that exhibits off targettoxicity in the context of an miRNA shuttle vector of the presentinvention limits the off target toxicity of the siRNA. In certainembodiments, the miRNA shuttle vector expresses a therapeutic siRNA inthe brain with limited off target toxicity. In certain embodiments, themiRNA shuttle vector expresses a therapeutic siRNA in the striatum withlimited off target toxicity. In certain embodiments, the miRNA shuttlevector expresses a therapeutic siRNA in the cerebrum with limited offtarget toxicity.

The present invention provides an isolated nucleic acid encoding aprimary transcript (pri-miRNA) including, in order of position, a5′-flanking region, a non-guide (passenger) region, a loop region, aguide region, and a 3′-flanking region, wherein the guide region is atleast 90% identical to CGACCAUGCGAGCCAGCA (miHDS.1 guide, SEQ ID NO:7),AGUCGCUGAUGACCGGGA (miHDS.2 guide, SEQ ID NO:8) or ACGUCGUAAACAAGAGGA(miHDS.5 guide, SEQ ID NO:9) and the non-guide region is at least 80%complementary to the guide region. In certain embodiments, the5′-flanking region is contiguously linked to the non-guide region, theloop region is positioned between the non-guide region and the guideregion, and the guide region is contiguously linked to the 3′-flankingregion. As used herein, the term “siRNA guide region” is asingle-stranded sequence of RNA that is complementary to a targetsequence. As used herein, the term “siRNA non-guide region” is asingle-stranded sequence of RNA that is complementary to the “siRNAguide region.” Thus, under the proper conditions, the siRNA guide regionand the siRNA non-guide region associate to form an RNA duplex. As usedherein, all nucleic acid sequences are listed, as is customary, in a 5′to 3′ direction.

In certain embodiments, the non-guide region is about 15-30 nucleotidesin length, and is about 70-100% complementary to the guide region, whichis about 15-30 nucleotides in length. In certain embodiments, the guideregion is at least 90% identical to CGACCAUGCGAGCCAGCA (miHDS.1 guide,SEQ ID NO:7), AGUCGCUGAUGACCGGGA (miHDS.2 guide, SEQ ID NO:8) orACGUCGUAAACAAGAGGA (miHDS.5 guide, SEQ ID NO:9) and the non-guide regionis at least 80% complementary to the guide region.

In certain embodiments, the 5′-flanking region contains a 5′-joiningsequence contiguously linked to the non-guide region. As used herein,the term “joining site” or a “joining sequence” is a short nucleic acidsequence of less than 60 nucleotides that connects two other nucleicacid sequences. In certain embodiments, the joining site is of a lengthof any integer between 4 and 50, inclusive. In certain embodiments, the5′-joining sequence consists of 5-8 nucleotides (e.g., consists of 6nucleotides). In certain embodiments, the 5′-joining sequence encodesGUGAGCGA (SEQ ID NO:12) or GUGAGCGC (SEQ ID NO:13).

In certain embodiments, the 5′-flanking region further comprises a5′-bulge sequence positioned upstream from the 5′-joining sequence. Asused herein, the term “bulge sequence” is a region of nucleic acid thatis non-complementary to the nucleic acid opposite it in a duplex. Forexample, a duplex will contain a region of complementary nucleic acids,then a region of non-complementary nucleic acids, followed by a secondregion of complementary nucleic acids. The regions of complementarynucleic acids will bind to each other, whereas the centralnon-complementary region will not bind, thereby forming a “bulge.” Incertain embodiments the two strands of nucleic acid positioned betweenthe two complementary regions will be of different lengths, therebyforming a “bulge.” In certain embodiments, the 5′-bulge sequence willcontain from 2 to 15 nucleotides. In certain embodiments, the 5′-bulgesequence consists of about 1-10 nucleotides. In certain embodiments, the5′-bulge sequence encodes UAAACUCGA (SEQ ID NO:14). In certainembodiments, the 5′-bulge sequence has from 0-50% complementarity to the3′-bulge sequence. The XhoI restriction site is CTCGAG (SEQ ID NO:15)(with “T” being “U” in RNA form in this and all other sequences listedherein).

In certain embodiments, the 5′-flanking region further contains a5′-spacer sequence positioned upstream from the 5′-bulge sequence. Incertain embodiments, the 5′-spacer sequence consists of 9-12nucleotides, such as 10-12 nucleotides. In certain embodiments, the5′-spacer sequence has from 60-100% complementarity to a 3′-spacersequence. In certain embodiments, the 5′-bulge sequence comprises acloning site, such as an XhoI site. In certain embodiments, the5′-spacer sequence is UGGUACCGUU (SEQ ID NO:16).

In certain embodiments, the 5′-flanking region further contains a5′-upstream sequence positioned upstream from the 5′-spacer sequence. Incertain embodiments, the 5′-upstream sequence is about 5-5000nucleotides in length, such as 30-2000 nucleotides in length.

In certain embodiments, the 3′-flanking region contains a 3′-joiningsequence contiguously linked to the guide region. In certainembodiments, the joining site is of a length of any integer between 4and 50, inclusive. In certain embodiments, the 3′-joining sequenceconsists of 5-8 nucleotides, (e.g., consists of 6 nucleotides). Incertain embodiments, the 3′-joining sequence is at least about 85%complementary to a 5′-joining sequence. In certain embodiments, the3′-joining sequence encodes CGCYUAC (SEQ ID NO:17), wherein Y is C or U.In certain embodiments, the 3′-joining sequence encodes CGCCUAC (SEQ IDNO:18).

In certain embodiments, the 3′-flanking region further comprises a3′-bulge sequence positioned downstream from the 3′-joining sequence. Incertain embodiments, the 3′-bulge sequence comprises a cloning site,such as a SpeI/XbaI site or a SpeI site. The SpeI/XbaI site is encodedby CTCAGA (SEQ ID NO:19), and the SpeI site is encoded by CTCAGT (SEQ IDNO:20). In certain embodiments, the 3′-bulge sequence consists of about1-15 nucleotides (such as 2-15 nucleotides or 1-10 nucleotides). Incertain embodiments, the 3′-bulge sequence encodes UAG (SEQ ID NO: 32).In certain embodiments, the 5′-bulge sequence is complementary to the3′-bulge sequence at only one nucleotide at each end of the sequence.

In certain embodiments, the 3′-flanking region further contains a3′-spacer sequence positioned downstream from the 3′-bulge sequence. Incertain embodiments, the 3′-spacer sequence consists of 9-12nucleotides, such as 10-12 nucleotides. In certain embodiments, the3′-spacer sequence is AGCGGCCGCCA (SEQ ID NO:21). In certainembodiments, the 3′-spacer sequence is at least about 70% complementaryto a 5′-spacer sequence.

In certain embodiments, the 3′-flanking region further contains a3′-downstream sequence positioned downstream from the 3′-spacersequence. In certain embodiments, a 5′-upstream sequence does notsignificantly pair with the 3′-downstream sequence. As used herein, theterm “does not significantly pair with” means that the two strands areless than 20% homologous. In certain embodiments, the 3′-downstreamsequence is about 5-5000 nucleotides in length, such as 30-2000nucleotides in length.

In certain embodiments, the loop region is from 4-20 nucleotides inlength, such as 15-19 nucleotides in length. From 0-50% of the loopregion can be complementary to another portion of the loop region. Asused herein, the term “loop region” is a sequence that joins twocomplementary strands of nucleic acid. In certain embodiments, 1-3nucleotides of the loop region are immediately contiguous to thecomplementary strands of nucleic acid may be complementary to the last1-3 nucleotides of the loop region. For example, the first two nucleicacids in the loop region may be complementary to the last twonucleotides of the loop region. In certain embodiments, the loop regionis 17 nucleotides in length. In certain embodiments, the loop regionencodes CUNNNNNNNNNNNNNNNGG (SEQ ID NO:22) or CCNNNNNNNNNNNNNNNGG (SEQID NO:23). In certain embodiments, the loop region encodesCUGUGAAGCCACAGAUGGG (SEQ ID NO:24) or CCGUGAAGCCACAGAUGGG (SEQ IDNO:25).

The present invention further provides an RNA encoded by nucleic aciddescribed herein.

The present invention further provides an expression cassette containinga promoter contiguously linked to a nucleic acid described herein. Incertain embodiments, the promoter is a polII or a polIII promoter, suchas a U6 promoter (e.g., a mouse U6 promoter). In certain embodiments,the expression cassette further contains a marker gene. In certainembodiments, the promoter is a polII promoter. In certain embodiments,the promoter is a tissue-specific promoter. In certain embodiments, thepromoter is an inducible promoter. In certain embodiments, the promoteris a polIII promoter.

The present invention provides a vector containing an expressioncassette described herein. In certain embodiments, the vector is anadeno-associated virus (AAV) vector.

The present invention provides a non-human animal comprising the nucleicacid, the expression cassette, or the vector described herein.

The present invention provides an isolated nucleic acid between 80-4000nucleotides in length comprising (or consisting of) an miHDS.1 guideGUCGACCAUGCGAGCCAGCAC (SEQ ID NO:4); an miHDS.2 guideAUAGUCGCUGAUGACCGGGAU (SEQ ID NO:5); an miHDS.5 guideUUACGUCGUAAACAAGAGGAA (SEQ ID NO:6); an miHDS.1CUCGAGUGAGCGAUGCUGGCUCGCAUGGUCGAUACUGUAAAGCCACAGAUGGGUGUCGACCAUGCGAGCCAGCACCGCCUACUAGA (SEQ ID NO:1),GCGUUUAGUGAACCGUCAGAUGGUACCGUUUAAACUCGAGUGAGCGAUGCUGGCUCGCAUGGUCGAUACUGUAAAGCCACAGAUGGGUGUCGACCAUGCGAGCCAGCACCGCCUACUAGAGCGGCCGCCACAGCGGGGAGAUCCAGACAUGAUAAGAU ACAUU (SEQ IDNO:10), or CUCGAGUGAGCGAUGCUGGCUCGCAUGGUCGAUACUGUAAAGCCACAGAUGGGUGUCGACCAUGCGAGCCAGCACCGCCUACUAGA (SEQ ID NO:33); an miHDS.2CUCGAGUGAGCGCUCCCGGUCAUCAGCGACUAUUCCGUAAAGCCACAGAUGGGGAUAGUCGCUGAUGACCGGGAUCGCCUACUAG (SEQ ID NO:2) orGCGUUUAGUGAACCGUCAGAUGGUACCGUUUAAACUCGAGUGAGCGCUCCCGGUCAUCAGCGACUAUUCCGUAAAGCCACAGAUGGGGAUAGUCGCUGAUGACCGGGAUCGCCUACUAGAGCGGCCGCCACAGCGGGGAGAUCCAGACAUGAUAAGAUA CAUU (SEQ IDNO:11); or an miHDS.5CUCGAGUGAGCGCUCCUCUUGUUUACGACGUGAUCUGUAAAGCCACAGAUGGGAUUACGUCGUAAACAAGAGGAACGCCUACUAGU (SEQ ID NO:3).

The present invention provides an isolated RNA duplex comprising a guideregion of nucleic acid and a non-guide region of nucleic acid, whereinthe guide region is at least 90% identical to CGACCAUGCGAGCCAGCA(miHDS.1 guide, SEQ ID NO:7), AGUCGCUGAUGACCGGGA (miHDS.2 guide, SEQ IDNO:8) or ACGUCGUAAACAAGAGGA (miHDS.5 guide, SEQ ID NO:9) and thenon-guide region is at least 80% complementary to the guide region. Incertain embodiments, the isolated RNA duplex is between 19-30 base pairsin length. Certain embodiments include an expression cassette encodingthe isolated nucleic acid described above. In certain embodiments theexpression cassette further comprises a marker gene.

The present invention provides method of inducing RNA interference byadministering to a subject a nucleic acid, an expression cassette, avector, or a composition described herein.

The present invention provides a vector containing a U6 promoteroperably linked to a nucleic acid encoding an miRNA. The predictedtranscription start sites of constructs of the present invention aredifferent from those used by researchers in the past. In certainembodiments of the present invention, the U6miRNA has an extended 5′end. If the 5′ end is truncated to resemble the previous CMV-basedstrategy, silencing efficacy is severely reduced. The present inventionalso provides improved flanking sequences that show improved efficacyover natural miR-30 flanking sequences. The use of the present miRNAstrategy appears to alleviate toxicity associated with traditional shRNAapproaches. The miRNA strategy does not generally generate excessiveamounts of RNAi as do U6shRNA approaches.

As used herein the term “stem sequence” is a sequence that iscomplementary to another sequence in the same molecule, where the twocomplementary strands anneal to form a duplex (e.g., the non-guide andguide regions). The duplex that is formed may be fully complementary, ormay be less than fully complementary, such as 99%, 98%, 97%, 96%, 95,%,94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%,80%, 75%, or 70% complementary to each other. Further, in certainembodiments, one strand may contain more nucleotides than the otherstrand, allowing the formation of a side loop.

The present invention also provides vectors containing the expressioncassettes described herein. Examples of appropriate vectors includeadenoviral, lentiviral, adeno-associated viral (AAV), poliovirus, herpessimplex virus (HSV), or murine Maloney-based viral vectors. In oneembodiment, the vector is an adeno-associated virus vector. Thesecassettes and vectors may be contained in a cell, such as a mammaliancell. A non-human mammal may contain the cassette or vector.

The present invention provides cells (such as a mammalian cell)containing the nucleic acid molecules, expression cassettes or vectorsdescribed herein. The present invention also provides a non-human mammalcontaining the nucleic acid molecules, expression cassettes or vectorsdescribed herein.

The present invention provides a nucleic acid, an expression cassette, avector, or a composition as described herein for use in therapy, such asfor treating a neurodegenerative disease.

The present invention provides an isolated RNAi molecule having amicroRNA having an overhang at the 3′ end. In certain embodiments, theoverhang is a 2 to 5-nucleotide repeat. In certain embodiments, theoverhang is a UU (SEQ ID NO:26), UUU (SEQ ID NO:27), UUUU (SEQ IDNO:28), CUU (SEQ ID NO:29), CUUU (SEQ ID NO:30) or CUUUU (SEQ ID NO:31)sequence. In certain embodiments, the microRNA is a naturally-occurringmicroRNA. In certain embodiments, microRNA is an artificial microRNA. Incertain embodiments, the RNAi molecule produces a decreased level ofoff-target toxicity.

The present invention provides a method of inducing low-toxicity RNAinterference by administering to a subject a nucleic acid, an expressioncassette, a vector, or a composition as described herein. In certainembodiments, the expression cassette contains a polII promoter.

The present invention provides a method of inducing low-toxicity RNAinterference by administering to a subject an expression cassetteencoding a polII promoter operably linked to a nucleic acid encoding amiRNA. In certain embodiments, the miRNA comprises a 2- or 3-nucleotide5′ or 3′-overhang. In certain embodiments, the miRNA comprises a2-nucleotide 3′-overhang. In certain embodiments, the miRNA is anartificial miRNA.

The present invention provides a method of treating a subject with aHuntington's Disease by administering to the subject a nucleic acid, anexpression cassette, a vector, or a composition as described herein soas to treat the Huntington's Disease.

The present invention provides a method of suppressing the accumulationof huntingtin in a cell by introducing nucleic acid molecules (e.g., aribonucleic acid (RNA)) described herein into the cell in an amountsufficient to suppress accumulation of huntingtin in the cell. Incertain embodiments, the accumulation of huntingtin is suppressed by atleast 10%. In certain embodiments, the accumulation of huntingtin issuppressed by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%,or 99%. In certain embodiments, the suppression of the accumulation ofthe protein is in an amount sufficient to cause a therapeutic effect,e.g., to reduce the formation of tangles.

The present invention provides a method of preventing cytotoxic effectsof mutant huntingtin in a cell by introducing nucleic acid molecules(e.g., a ribonucleic acid (RNA)) described herein into the cell in anamount sufficient to suppress accumulation of huntingtin. In certainembodiments, the nucleic acid molecules prevents cytotoxic effects ofhuntingtin, e.g., in a neuronal cell.

The present invention provides a method to inhibit expression of ahuntingtin gene in a cell by introducing a nucleic acid molecule (e.g.,a ribonucleic acid (RNA)) described herein into the cell in an amountsufficient to inhibit expression of the huntingtin, and wherein the RNAinhibits expression of the huntingtin gene. In certain embodiments, thehuntingtin is inhibited by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90% 95%, or 99%.

The present invention provides a method to inhibit expression of ahuntingtin gene in a mammal (e.g., a human or a non-human mammal) by (a)providing a mammal containing a neuronal cell, wherein the neuronal cellcontains the huntingtin gene and the neuronal cell is susceptible to RNAinterference, and the huntingtin gene is expressed in the neuronal cell;and (b) contacting the mammal with a ribonucleic acid (RNA) or a vectordescribed herein, thereby inhibiting expression of the huntingtin gene.In certain embodiments, the accumulation of huntingtin is suppressed byat least 10%. In certain embodiments, the huntingtin is inhibited by atleast 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99%. Incertain embodiments, the cell is located in vivo in a mammal.

The present invention provides a viral vector comprising a promoter anda micro RNA (miRNA) shuttle containing an embedded siRNA specific for atarget sequence. In certain embodiments, the promoter is an induciblepromoter. In certain embodiments, the vector is an adenoviral,lentiviral, adeno-associated viral (AAV), poliovirus, HSV, or murineMaloney-based viral vector. In certain embodiments, the targetedsequence is a sequence associated with Huntington's Disease. The targetsequence, in certain embodiments, is a sequence encoding huntingtin.

The present invention provides a method of preventing cytotoxic effectsof neurodegenerative disease in a mammal in need thereof, by introducingthe vector encoding a miRNA described herein into a cell in an amountsufficient to suppress accumulation of a protein associated withHuntington's Disease, and wherein the RNA prevents cytotoxic effects ofHuntington's Disease (also referred to as HD, and the protein involvedis huntingtin, also called htt).

The present invention also provides a method to inhibit expression of aprotein associated with Huntington's Disease in a mammal in needthereof, by introducing the vector encoding a miRNA described hereininto a cell in an amount sufficient to inhibit expression of thehuntingtin protein, wherein the RNA inhibits expression of thehuntingtin protein. The huntingtin protein is inhibited by at least 10%,20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% 95%, or 99%.

This invention relates to compounds, compositions, and methods usefulfor modulating Huntington's Disease gene expression using shortinterfering nucleic acid (siRNA) molecules. This invention also relatesto compounds, compositions, and methods useful for modulating theexpression and activity of other genes involved in pathways of HD geneexpression and/or activity by RNA interference (RNAi) using smallnucleic acid molecules. In particular, the instant invention featuressmall nucleic acid molecules, such as short interfering nucleic acid(siNA), short interfering RNA (siRNA), double-stranded RNA (dsRNA),micro-RNA (miRNA), and short hairpin RNA (shRNA) molecules and methodsused to modulate the expression HD genes. A siRNA molecule of theinstant invention can be, e.g., chemically synthesized, expressed from avector or enzymatically synthesized.

As used herein when a claim indicates an RNA “corresponding to” it ismeant the RNA that has the same sequence as the DNA, except that uracilis substituted for thymine.

The present invention further provides a method of substantiallysilencing a target gene of interest or targeted allele for the gene ofinterest in order to provide a therapeutic effect. As used herein theterm “substantially silencing” or “substantially silenced” refers todecreasing, reducing, or inhibiting the expression of the target gene ortarget allele by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% to 100%. As used herein theterm “therapeutic effect” refers to a change in the associatedabnormalities of the disease state, including pathological andbehavioral deficits; a change in the time to progression of the diseasestate; a reduction, lessening, or alteration of a symptom of thedisease; or an improvement in the quality of life of the personafflicted with the disease. Therapeutic effects can be measuredquantitatively by a physician or qualitatively by a patient afflictedwith the disease state targeted by the siRNA. In certain embodimentswherein both the mutant and wild type allele are substantially silenced,the term therapeutic effect defines a condition in which silencing ofthe wild type allele's expression does not have a deleterious or harmfuleffect on normal functions such that the patient would not have atherapeutic effect.

In one embodiment, the invention features a method for treating orpreventing Huntington's Disease in a subject or organism comprisingcontacting the subject or organism with a siRNA of the invention underconditions suitable to modulate the expression of the HD gene in thesubject or organism whereby the treatment or prevention of Huntington'sDisease can be achieved. In one embodiment, the HD gene target comprisesboth HD allele (e.g., an allele comprising a trinucleotide (CAG) repeatexpansion and a wild type allele). The siRNA molecule of the inventioncan be expressed from vectors as described herein or otherwise known inthe art to target appropriate tissues or cells in the subject ororganism.

In one embodiment, the invention features a method for treating orpreventing Huntington's Disease in a subject or organism comprising,contacting the subject or organism with a siRNA molecule of theinvention via local administration to relevant tissues or cells, such asbrain cells and tissues (e.g., basal ganglia, striatum, or cortex), forexample, by administration of vectors or expression cassettes of theinvention that provide siRNA molecules of the invention to relevantcells (e.g., basal ganglia, striatum, or cortex). In one embodiment, thesiRNA, vector, or expression cassette is administered to the subject ororganism by stereotactic or convection enhanced delivery to the brain.For example, U.S. Pat. No. 5,720,720 provides methods and devices usefulfor stereotactic and convection enhanced delivery of reagents to thebrain. Such methods and devices can be readily used for the delivery ofsiRNAs, vectors, or expression cassettes of the invention to a subjector organism, and is U.S. Pat. No. 5,720,720 is incorporated by referenceherein in its entirety. US Patent Application Nos. 2002/0141980;2002/0114780; and 2002/0187127 all provide methods and devices usefulfor stereotactic and convection enhanced delivery of reagents that canbe readily adapted for delivery of siRNAs, vectors, or expressioncassettes of the invention to a subject or organism, and areincorporated by reference herein in their entirety. Particular devicesthat may be useful in delivering siRNAs, vectors, or expressioncassettes of the invention to a subject or organism are for exampledescribed in US Patent Application No. 2004/0162255, which isincorporated by reference herein in its entirety. The siRNA molecule ofthe invention can be expressed from vectors as described herein orotherwise known in the art to target appropriate tissues or cells in thesubject or organism.

Methods of delivery of viral vectors include, but are not limited to,intra-arterial, intra-muscular, intravenous, intranasal and oral routes.Generally, AAV virions may be introduced into cells of the CNS usingeither in vivo or in vitro transduction techniques. If transduced invitro, the desired recipient cell will be removed from the subject,transduced with AAV virions and reintroduced into the subject.Alternatively, syngeneic or xenogeneic cells can be used where thosecells will not generate an inappropriate immune response in the subject.

Suitable methods for the delivery and introduction of transduced cellsinto a subject have been described. For example, cells can be transducedin vitro by combining recombinant AAV virions with CNS cells e.g., inappropriate media, and screening for those cells harboring the DNA ofinterest can be screened using conventional techniques such as Southernblots and/or PCR, or by using selectable markers. Transduced cells canthen be formulated into pharmaceutical compositions, described morefully below, and the composition introduced into the subject by varioustechniques, such as by grafting, intramuscular, intravenous,subcutaneous and intraperitoneal injection.

In one embodiment, for in vivo delivery, AAV virions are formulated intopharmaceutical compositions and will generally be administeredparenterally, e.g., by intramuscular injection directly into skeletal orcardiac muscle or by injection into the CNS.

In one embodiment, viral vectors of the invention are delivered to theCNS via convection-enhanced delivery (CED) systems that can efficientlydeliver viral vectors, e.g., AAV, over large regions of a subject'sbrain (e.g., striatum and/or cortex). As described in detail andexemplified below, these methods are suitable for a variety of viralvectors, for instance AAV vectors carrying therapeutic genes (e.g.,siRNAs).

Any convection-enhanced delivery device may be appropriate for deliveryof viral vectors. In one embodiment, the device is an osmotic pump or aninfusion pump. Both osmotic and infusion pumps are commerciallyavailable from a variety of suppliers, for example Alzet Corporation,Hamilton Corporation, Aiza, Inc., Palo Alto, Calif.). Typically, a viralvector is delivered via CED devices as follows. A catheter, cannula orother injection device is inserted into CNS tissue in the chosensubject. In view of the teachings herein, one of skill in the art couldreadily determine which general area of the CNS is an appropriatetarget. For example, when delivering AAV vector encoding a therapeuticgene to treat HD, the striatum is a suitable area of the brain totarget. Stereotactic maps and positioning devices are available, forexample from ASI Instruments, Warren, Mich. Positioning may also beconducted by using anatomical maps obtained by CT and/or MRI imaging ofthe subject's brain to help guide the injection device to the chosentarget. Moreover, because the methods described herein can be practicedsuch that relatively large areas of the brain take up the viral vectors,fewer infusion cannula are needed. Since surgical complications arerelated to the number of penetrations, the methods described herein alsoserve to reduce the side effects seen with conventional deliverytechniques.

In one embodiment, pharmaceutical compositions will comprise sufficientgenetic material to produce a therapeutically effective amount of thesiRNA of interest, i.e., an amount sufficient to reduce or amelioratesymptoms of the disease state in question or an amount sufficient toconfer the desired benefit. The pharmaceutical compositions may alsocontain a pharmaceutically acceptable excipient. Such excipients includeany pharmaceutical agent that does not itself induce the production ofantibodies harmful to the individual receiving the composition, andwhich may be administered without undue toxicity. Pharmaceuticallyacceptable excipients include, but are not limited to, sorbitol,Tween80, and liquids such as water, saline, glycerol and ethanol.Pharmaceutically acceptable salts can be included therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles. A thoroughdiscussion of pharmaceutically acceptable excipients is available inREMINGTON'S PHARMACEUTICAL SCIENCES (Mack Pub. Co., N.J. 1991).

As is apparent to those skilled in the art in view of the teachings ofthis specification, an effective amount of viral vector which must beadded can be empirically determined. Administration can be effected inone dose, continuously or intermittently throughout the course oftreatment. Methods of determining the most effective means and dosagesof administration are well known to those of skill in the art and willvary with the viral vector, the composition of the therapy, the targetcells, and the subject being treated. Single and multipleadministrations can be carried out with the dose level and pattern beingselected by the treating physician.

It should be understood that more than one transgene could be expressedby the delivered viral vector. Alternatively, separate vectors, eachexpressing one or more different transgenes, can also be delivered tothe CNS as described herein. Furthermore, it is also intended that theviral vectors delivered by the methods of the present invention becombined with other suitable compositions and therapies.

The present invention further provides an miRNA or shRNA, an expressioncassette and/or a vector as described herein for use in medicaltreatment or diagnosis.

The present invention provides the use of an miRNA or shRNA, anexpression cassette and/or a vector as described herein to prepare amedicament useful for treating a condition amenable to RNAi in ananimal, e.g., useful for treating Huntington's Disease.

The present invention also provides a nucleic acid, expression cassette,vector, or composition of the invention for use in therapy.

The present invention also provides a nucleic acid, expression cassette,vector, or composition of the invention for treating, e.g., for use inthe prophylactic or therapeutic treatment of, Huntington's Disease.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. The artificial miRNA, miSCR, causes neurotoxicity in mousebrain. Wild-type mice were injected into the striatum with AAV-GFP(expresses GFP only) or AAV-miSCR-GFP (expresses both the artificialmiRNA and GFP), and histological analyses were performed on brainsharvested at 6 months post-treatment. Photomicrographs representing GFPautofluorescence and immunohistochemical staining of IbaI-positivemicroglia are shown. Scale bars=200 and 50 μm for 10× and 40× imagesrespectively.

FIG. 2. Overview of seed-related off-targeting: mechanism andprobabilities. (a) Diagram depicting the expression and processing of anartificial miRNA (SEQ ID NO: 203) to produce the mature siRNA duplex(SEQ ID NOs: 204 and 205, respectively, in order of appearance). Theantisense guide strand is loaded into RISC and may direct on-targetsilencing (intended) (SEQ ID NOs: 205 and 206, respectively, in order ofappearance) and off-target silencing (unintended) (SEQ ID NOs: 205 and207, respectively, in order of appearance). (b) Cartoon highlighting therelationship between the frequencies of seed complement binding sites inthe 3′-UTRome and the off-targeting potential for siRNAs. (c) The numberof human mRNA 3′-UTRs containing a given hexamer was determined for allof the 4096 possible hexamers and a binned distribution is shown. Theprobabilities that randomly selected siRNAs targeting human codingsequence (CDS) will contain seed complements in a given range (white andgrey shading) are also presented. For example, there is only a 10%chance that a randomly selected siRNA contains a seed complement for ahexamer present in ˜1500 human 3′-UTRs or less. Note: the sequencestested in this manuscript are placed above their respective ranges.

FIG. 3. Selection and screening of htt-targeting siRNAs with lowoff-targeting potentials. (a) Schematic outlining the selection of“safe” seed siRNAs with proper strand-biasing. FIG. 3a discloses SEQ IDNOs: 208-210, respectively, in order of appearance. (b) Plasmidsexpressing artificial miRNAs, harboring the indicated siRNA sequences,were transfected into HEK293 cells, and QPCR analysis was performed 24 hlater to measure endogenous htt mRNA levels. U6 (promoter-only) andHD2.4 (a previously published htt RNAi sequence) serve as the negativeand positive controls respectively. Results are shown as mean±SEM(N=6, * indicates P<0.001, relative to U6).

FIG. 4. Evaluation of microarray data for htt silencing andoff-targeting. HEK293 cells were transfected with U6 promoter-only orU6-driven artificial miRNA expression plasmids (n=4 for each treatment),and RNA was harvested 72 h later for microarray analysis. Two-way ANOVAwas performed to detect differentially expressed genes among thetreatment groups. (a) Htt mRNA levels determined by microarray (greybars) were consistent with those measured by QPCR (black bars) using thesame RNA samples. (b) Hierarchical clustering and heat-maps weregenerated using differentially expressed genes (P<0.0001, 825 genes) tovisualize the relationships among the treatment groups. Interestingly,all of the “safe” seed sequences are more related to U6 than theremaining sequences predicted to have higher off-targeting potentials(boundary marked by white line). (c) Hierarchical clustering andheat-maps were generated using differentially expressed genes (P<0.01,992 genes) to visualize the relationships among the treatment groups.The impact of seed sequence on gene expression can be appreciated by theclustering of 8.2 and 8.2 mis which share the same seed. Notably, thepredicted low off-targeting sequences (Safe, HDS1 and HDS2) are moresimilar to U6, and have smaller off-targeting signatures compared toboth 2.4 and 8.2. Seed-related off-targeting was evaluated by cumulativedistribution (d) and motif discovery (e) analyses. (d) Cumulativedistribution plots for gene expression values are shown for transcriptscontaining (1 site or 2+ sites) or lacking (baseline) 3′-UTR seedcomplement binding sites for the indicated sequence and strand. A shiftto the left indicates an increased likelihood of being down-regulated.AS=antisense, S=sense. KS-test P-values are shown; N.S.=no statisticalsignificance (P>0.1). (e) Motif discovery analyses identified anenrichment of seed complement binding sites in the 3′-UTRs ofdown-regulated genes (>1.1-fold) unique to each treatment. Shown hereare the examples of 8.2-124a (SEQ ID NO: 212) and Terror (SEQ ID NO:211); similar data for the remaining sequences supports that eachmediates detectable seed-related off-targeting to some degree (see FIG.6 below).

FIG. 5. Silencing efficacy and safety of HDS sequences in mouse brain.Wild-type mice were injected into the striatum with AAV virusesco-expressing artificial miRNAs and GFP. (a) At 3 weeks post-injection,GFP-positive striata were harvested and QPCR analysis was performed tomeasure endogenous mouse Htt mRNA levels. Results are shown as mean±SEM(n≧3, * indicates P=0.001, relative to uninjected striata). (b) Brainsfrom additional cohorts of injected mice were harvested at 6 monthspost-injection and histological analyses were performed to assessneurotoxicity. Photomicrographs representing GFP autofluorescence andimmunohistochemical staining of IbaI-positive microglia are shown. Scalebars=200 and 50 μm for 10× and 40× images respectively.

FIG. 6. Evaluation of microarray data for off-targeting. Seed-relatedoff-targeting was evaluated by cumulative distribution (a) and motifdiscovery (b) analyses. (a) Cumulative distribution plots for geneexpression values are shown for transcripts containing (1 site or 2+sites) or lacking (baseline) 3′-UTR seed complement binding sites forthe indicated sequence and strand. A shift to the left indicates anincreased likelihood of being down-regulated. AS=antisense. KS-testP-values are shown; N.S.=no statistical significance (P>0.1). (b) Motifdiscovery analyses identified an enrichment of seed complement bindingsites in the 3′-UTRs of down-regulated genes (>1.1-fold) unique to eachtreatment (SEQ ID NOs: 210 and 213-217, respectively, in order ofappearance).

FIG. 7. Full-length sequences and structures for pri-miHDS.1. FIG. 7discloses SEQ ID NOs: 10, 218, 219 and 210, respectively, in order ofappearance.

FIG. 8. Full-length sequences and structures for pri-miHDS.2. FIG. 8discloses SEQ ID NOs: 11, 220, 221 and 213, respectively, in order ofappearance.

DETAILED DESCRIPTION OF THE INVENTION

RNA Interference (RNAi) is a process of gene regulation mediated bysmall dsRNAs. RNAi is used as a common biological tool to study genefunction, and is under investigation as a therapeutic to treat variousdiseases. RNAi delivery or expression can be through the administrationof exogenous siRNAs (transient gene silencing) or through theadministration of vectors expressing stem-loop RNAs (persistent genesilencing). The absolute specificity of RNAi is questionable. Issuesthat must be addressed include cellular responses to dsRNA (IFN-b, PKR,OAS1) and off-target effects due to saturation of RNAi machinery or viapartial complementarity with unintended mRNAs. There is an on-going needfor optimizing RNAi vectors and potentially developing tissue-specificand regulated expression strategies

The use of RNAi as a therapeutic is dependant upon the elucidation ofseveral factors including i) the delivery and persistence of the RNAiconstruct for effective silencing of the target gene sequence; ii) thedesign of the siRNA in order to achieve effective knock down or genesuppression of the target sequence, and iii) the optimal siRNAexpression system (shRNA or miRNA) for delivery of the therapeuticsiRNA. While many studies have evaluated the use of RNAi delivered aschemically synthesized oligonucleotide structures, for many clinicalconditions and disease states such as Huntington's Disease, it isbelieved that to achieve therapeutic benefit there is a need for longterm and or persistent high level expression of the therapeutic siRNA asachieved by endogenous production of expressed siRNA. To date, shRNA-and artificial miRNA-based strategies have been compared withconflicting results. The therapeutic utility of expressed RNAi isunresolved due to safety concerns as a result of off target toxicityarising from cellular responses to dsRNA (IFN-b, PKR, OAS1), saturationof RNAi machinery or silencing of off targets via partialcomplementarity with unintended mRNAs. Thus, there is an on-going needfor optimizing expressed RNAi vectors that are safe and effective.

shRNAs are comprised of stem-loop structures which are designed tocontain a 5′ flanking region, siRNA region segments, a loop region, a 3′siRNA region and a 3′ flanking region. Most RNAi expression strategieshave utilized short-hairpin RNAs (shRNAs) driven by strong polIII-basedpromoters. Many shRNAs have demonstrated effective knock down of thetarget sequences in vitro as well as in vivo, however, some shRNAs whichdemonstrated effective knock down of the target gene were also found tohave toxicity in vivo. A recently discovered alternative approach is theuse of artificial miRNAs (pri-miRNA scaffolds shuttling siRNA sequences)as RNAi vectors. Artificial miRNAs more naturally resemble endogenousRNAi substrates and are more amenable to Pol-II transcription (e.g.,allowing tissue-specific expression of RNAi) and polycistronicstrategies (e.g., allowing delivery of multiple siRNA sequences). Todate the efficacy of miRNA based vector systems compared to shRNA hasbeen confounded by conflicting results. Importantly, the question ofoff-target toxicity produced by the two systems has not been evaluated.

An important consideration for development of expressed siRNA is theconcept of “dosing” the host cell with the expressed siRNA construct.“Dosing” for an expressed siRNA in the context of the present inventionrefers to and can be dependant on the delivery vehicle (e.g., viral ornonviral), the relative amounts or concentration of the deliveryvehicle, and the strength and specificity of the promoter utilized todrive the expression of the siRNA sequence.

The inventors have developed artificial miRNA shuttle vectors thatincorporate the stem loop sequences contained in shRNAs withinmodifications of a naturally occurring human microRNA 30 sequence ormi30 sequence that serve to shuttle these small interfering RNA (siRNA)sequences. See, e.g., PCT Publication WO 2008/150897, which isincorporated by reference herein.

MicroRNA Shuttles for RNAi

miRNAs are small cellular RNAs (˜22nt) that are processed from precursorstem loop transcripts. Known miRNA stem loops can be modified to containRNAi sequences specific for genes of interest. miRNA molecules can bepreferable over shRNA molecules because miRNAs are endogenouslyexpressed. Therefore, miRNA molecules are unlikely to inducedsRNA-responsive interferon pathways, they are processed moreefficiently than shRNAs, and they have been shown to silence 80% moreeffectively.

Also, the promoter roles are different for miRNA molecules as comparedto shRNA molecules. Tissue-specific, inducible expression of shRNAsinvolves truncation of polII promoters to the transcription start site.In contrast, miRNAs can be expressed from any polII promoter because thetranscription start and stop sites can be relatively arbitrary.

Treatment of Huntington's Disease

The dominant polyglutamine expansion diseases, which includeSpinocerebellar ataxia type 1 (SCA1) and Huntington's disease (HD), areprogressive, untreatable neurodegenerative disorders. In inducible mousemodels HD, repression of mutant allele expression improves diseasephenotypes. Thus, therapies designed to inhibit disease gene expressionwould be beneficial. The present invention provides methods of usingRNAi in vivo to treat Huntington's Disease. “Treating” as used hereinrefers to ameliorating at least one symptom of, curing and/or preventingthe development of a disease or a condition.

In certain embodiment of the invention, RNAi molecules are employed toinhibit expression of a target gene. By “inhibit expression” is meant toreduce, diminish or suppress expression of a target gene. Expression ofa target gene may be inhibited via “gene silencing.” Gene silencingrefers to the suppression of gene expression, e.g., transgene,heterologous gene and/or endogenous gene expression, which may bemediated through processes that affect transcription and/or throughprocesses that affect post-transcriptional mechanisms. In someembodiments, gene silencing occurs when an RNAi molecule initiates theinhibition or degradation of the mRNA transcribed from a gene ofinterest in a sequence-specific manner via RNA interference, therebypreventing translation of the gene's product.

The reference to siRNAs herein is meant to include shRNAs and othersmall RNAs that can or are capable of modulating the expression of atargeted gene, e.g., the HD gene, for example via RNA interference. Suchsmall RNAs include without limitation, shRNAs and miroRNAs (miRNAs).

Disclosed herein is a strategy that results in substantial silencing oftargeted genes via RNAi. Use of this strategy results in markedlydiminished in vitro and in vivo expression of targeted genes. Thisstrategy is useful in reducing expression of targeted genes in order tomodel biological processes or to provide therapy for human diseases. Forexample, this strategy can be applied to Huntington's Disease. As usedherein the term “substantial silencing” means that the mRNA of thetargeted gene is inhibited and/or degraded by the presence of theintroduced siRNA, such that expression of the targeted gene is reducedby about 10% to 100% as compared to the level of expression seen whenthe siRNA is not present. Generally, when an gene is substantiallysilenced, it will have at least 40%, 50%, 60%, to 70%, e.g., 71%, 72%,73%, 74%, 75%, 76%, 77%, 78%, to 79%, generally at least 80%, e.g.,81%-84%, at least 85%, e.g., 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or even 100% reduction expression ascompared to when the siRNA is not present. As used herein the term“substantially normal activity” means the level of expression of a genewhen an siRNA has not been introduced to a cell.

Huntington disease (HD) is a strong candidate for siRNA-based therapy.HD is caused by CAG repeat expansions that encode polyQ in the diseaseprotein. PolyQ expansion confers a dominant toxic property on the mutantprotein that is associated with aberrant accumulation of the diseaseprotein in neurons. HD is progressive, ultimately fatal disorders thattypically begin in adulthood. Expansion of the CAG repeat/polyQ domainconfers upon the encoded protein a dominant toxic property. Thus, as atherapeutic strategy, efforts to lower expression of the mutant geneproduct prior to cell death could be highly beneficial to patients.

RNA Interference (RNAi) Molecules

An “RNA interference,” “RNAi,” “small interfering RNA” or “shortinterfering RNA” or “siRNA” or “short hairpin RNA” or “shRNA” molecule,or “miRNA” is a RNA duplex of nucleotides that is targeted to a nucleicacid sequence of interest, for example, huntingtin (htt). As usedherein, the term “siRNA” is a generic term that encompasses the subsetof shRNAs and miRNAs. An “RNA duplex” refers to the structure formed bythe complementary pairing between two regions of a RNA molecule. siRNAis “targeted” to a gene in that the nucleotide sequence of the duplexportion of the siRNA is complementary to a nucleotide sequence of thetargeted gene. In certain embodiments, the siRNAs are targeted to thesequence encoding ataxin-1 or huntingtin. In some embodiments, thelength of the duplex of siRNAs is less than 30 base pairs. In someembodiments, the duplex can be 29, 28, 27, 26, 25, 24, 23, 22, 21, 20,19, 18, 17, 16, 15, 14, 13, 12, 11 or 10 base pairs in length. In someembodiments, the length of the duplex is 19 to 25 base pairs in length.In certain embodiment, the length of the duplex is 19 or 21 base pairsin length. The RNA duplex portion of the siRNA can be part of a hairpinstructure. In addition to the duplex portion, the hairpin structure maycontain a loop portion positioned between the two sequences that formthe duplex. The loop can vary in length. In some embodiments the loop is5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24 or 25 nucleotides in length. In certain embodiments, the loop is 18nucleotides in length. The hairpin structure can also contain 3′ and/or5′ overhang portions. In some embodiments, the overhang is a 3′ and/or a5′ overhang 0, 1, 2, 3, 4 or 5 nucleotides in length.

The transcriptional unit of a “shRNA” is comprised of sense andantisense sequences connected by a loop of unpaired nucleotides. shRNAsare exported from the nucleus by Exportin-5, and once in the cytoplasm,are processed by Dicer to generate functional siRNAs. “miRNAs”stem-loops are comprised of sense and antisense sequences connected by aloop of unpaired nucleotides typically expressed as part of largerprimary transcripts (pri-miRNAs), which are excised by the Drosha-DGCR8complex generating intermediates known as pre-miRNAs, which aresubsequently exported from the nucleus by Exportin-5, and once in thecytoplasm, are processed by Dicer to generate functional siRNAs.“Artificial miRNA” or an “artificial miRNA shuttle vector,” as usedherein interchangably, refers to a primary miRNA transcript that has hada region of the duplex stem loop (at least about 9-20 nucleotides) whichis excised via Drosha and Dicer processing replaced with the siRNAsequences for the target gene while retaining the structural elementswithin the stem loop necessary for effective Drosha processing. The term“artificial” arises from the fact the flanking sequences (˜35nucleotides upstream and ˜40 nucleotides downstream) arise fromrestriction enzyme sites within the multiple cloning site of the siRNA.As used herein the term “miRNA” encompasses both the naturally occurringmiRNA sequences as well as artificially generated miRNA shuttle vectors.

The siRNA can be encoded by a nucleic acid sequence, and the nucleicacid sequence can also include a promoter. The nucleic acid sequence canalso include a polyadenylation signal. In some embodiments, thepolyadenylation signal is a synthetic minimal polyadenylation signal ora sequence of six Ts.

“Off-target toxicity” refers to deleterious, undesirable, or unintendedphenotypic changes of a host cell that expresses or contains an siRNA.Off-target toxicity may result in loss of desirable function, gain ofnon-desirable function, or even death at the cellular or organismallevel. Off-target toxicity may occur immediately upon expression of thesiRNA or may occur gradually over time. Off-target toxicity may occur asa direct result of the expression siRNA or may occur as a result ofinduction of host immune response to the cell expressing the siRNA.Without wishing to be bound by theory, off-target toxicity is postulatedto arise from high levels or overabundance of RNAi substrates within thecell. These overabundant or overexpressed RNAi substrates, includingwithout limitation pre- or pri RNAi substrates as well as overabundantmature antisense-RNAs, may compete for endogenous RNAi machinery, thusdisrupting natural miRNA biogenesis and function. Off-target toxicitymay also arise from an increased likelihood of silencing of unintendedmRNAs (i.e., off-target) due to partial complementarity of the sequence.Off target toxicity may also occur from improper strand biasing of anon-guide region such that there is preferential loading of thenon-guide region over the targeted or guide region of the RNAi.Off-target toxicity may also arise from stimulation of cellularresponses to dsRNAs which include dsRNA (IFN-b, PKR, OAS1). “Decreasedoff target toxicity” refers to a decrease, reduction, abrogation orattenuation in off target toxicity such that the therapeutic effect ismore beneficial to the host than the toxicity is limiting or detrimentalas measured by an improved duration or quality of life or an improvedsign or symptom of a disease or condition being targeted by the siRNA.“Limited off target toxicity” or “low off target toxicity” is used torefer to an unintended undesirable phenotypic changes to a cell ororganism, whether detectable or not, that does not preclude or outweighor limit the therapeutic benefit to the host treated with the siRNA andmay be considered a “side effect” of the therapy. Decreased or limitedoff target toxicity may be determined or inferred by comparing the invitro analysis such as Northern blot or QPCR for the levels of siRNAsubstrates or the in vivo effects comparing an equivalent shRNA vectorto the miRNA shuttle vector of the present invention.

“Knock-down,” “knock-down technology” refers to a technique of genesilencing in which the expression of a target gene is reduced ascompared to the gene expression prior to the introduction of the siRNA,which can lead to the inhibition of production of the target geneproduct. The term “reduced” is used herein to indicate that the targetgene expression is lowered by 1-100%. In other words, the amount of RNAavailable for translation into a polypeptide or protein is minimized.For example, the amount of protein may be reduced by 10, 20, 30, 40, 50,60, 70, 80, 90, 95, or 99%. In some embodiments, the expression isreduced by about 90% (i.e., only about 10% of the amount of protein isobserved a cell as compared to a cell where siRNA molecules have notbeen administered). Knock-down of gene expression can be directed by theuse of dsRNAs or siRNAs.

“RNA interference (RNAi)” is the process of sequence-specific,post-transcriptional gene silencing initiated by siRNA. During RNAi,siRNA induces degradation of target mRNA with consequentsequence-specific inhibition of gene expression.

According to a method of the present invention, the expression ofhuntingtin can be modified via RNAi. For example, the accumulation ofhuntingtin can be suppressed in a cell. The term “suppressing” refers tothe diminution, reduction or elimination in the number or amount oftranscripts present in a particular cell. For example, the accumulationof mRNA encoding huntingtin can be suppressed in a cell by RNAinterference (RNAi), e.g., the gene is silenced by sequence-specificdouble-stranded RNA (dsRNA), which is also called short interfering RNA(siRNA). These siRNAs can be two separate RNA molecules that havehybridized together, or they may be a single hairpin wherein twoportions of a RNA molecule have hybridized together to form a duplex.

A mutant protein refers to the protein encoded by a gene having amutation, e.g., a missense or nonsense mutation in huntingtin. A mutanthuntingtin may be disease-causing, i.e., may lead to a diseaseassociated with the presence of huntingtin in an animal having eitherone or two mutant allele(s).

The term “gene” is used broadly to refer to any segment of nucleic acidassociated with a biological function. Thus, genes include codingsequences and/or the regulatory sequences required for their expression.For example, “gene” refers to a nucleic acid fragment that expressesmRNA, functional RNA, or specific protein, including regulatorysequences. “Genes” also include nonexpressed DNA segments that, forexample, form recognition sequences for other proteins. “Genes” can beobtained from a variety of sources, including cloning from a source ofinterest or synthesizing from known or predicted sequence information,and may include sequences designed to have desired parameters. An“allele” is one of several alternative forms of a gene occupying a givenlocus on a chromosome.

The term “nucleic acid” refers to deoxyribonucleic acid (DNA) orribonucleic acid (RNA) and polymers thereof in either single- ordouble-stranded form, composed of monomers (nucleotides) containing asugar, phosphate and a base that is either a purine or pyrimidine.Unless specifically limited, the term encompasses nucleic acidscontaining known analogs of natural nucleotides that have similarbinding properties as the reference nucleic acid and are metabolized ina manner similar to naturally occurring nucleotides. Unless otherwiseindicated, a particular nucleic acid sequence also encompassesconservatively modified variants thereof (e.g., degenerate codonsubstitutions) and complementary sequences, as well as the sequenceexplicitly indicated. Specifically, degenerate codon substitutions maybe achieved by generating sequences in which the third position of oneor more selected (or all) codons is substituted with mixed-base and/ordeoxyinosine residues. A “nucleic acid fragment” is a portion of a givennucleic acid molecule.

A “nucleotide sequence” is a polymer of DNA or RNA that can besingle-stranded or double-stranded, optionally containing synthetic,non-natural or altered nucleotide bases capable of incorporation intoDNA or RNA polymers.

The terms “nucleic acid,” “nucleic acid molecule,” “nucleic acidfragment,” “nucleic acid sequence or segment,” or “polynucleotide” areused interchangeably and may also be used interchangeably with gene,cDNA, DNA and RNA encoded by a gene.

The invention encompasses isolated or substantially purified nucleicacid nucleic acid molecules and compositions containing those molecules.In the context of the present invention, an “isolated” or “purified” DNAmolecule or RNA molecule is a DNA molecule or RNA molecule that existsapart from its native environment and is therefore not a product ofnature. An isolated DNA molecule or RNA molecule may exist in a purifiedform or may exist in a non-native environment such as, for example, atransgenic host cell. For example, an “isolated” or “purified” nucleicacid molecule or biologically active portion thereof, is substantiallyfree of other cellular material, or culture medium when produced byrecombinant techniques, or substantially free of chemical precursors orother chemicals when chemically synthesized. In one embodiment, an“isolated” nucleic acid is free of sequences that naturally flank thenucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For example, in various embodiments, the isolatednucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2kb, 1 kb, 0.5 kb, or 0.1 kb of nucleotide sequences that naturally flankthe nucleic acid molecule in genomic DNA of the cell from which thenucleic acid is derived. Fragments and variants of the disclosednucleotide sequences are also encompassed by the present invention. By“fragment” or “portion” is meant a full length or less than full lengthof the nucleotide sequence.

“Naturally occurring,” “native,” or “wild-type” is used to describe anobject that can be found in nature as distinct from being artificiallyproduced. For example, a protein or nucleotide sequence present in anorganism (including a virus), which can be isolated from a source innature and that has not been intentionally modified by a person in thelaboratory, is naturally occurring.

A “variant” of a molecule is a sequence that is substantially similar tothe sequence of the native molecule. For nucleotide sequences, variantsinclude those sequences that, because of the degeneracy of the geneticcode, encode the identical amino acid sequence of the native protein.Naturally occurring allelic variants such as these can be identifiedwith the use of molecular biology techniques, as, for example, withpolymerase chain reaction (PCR) and hybridization techniques. Variantnucleotide sequences also include synthetically derived nucleotidesequences, such as those generated, for example, by using site-directedmutagenesis, which encode the native protein, as well as those thatencode a polypeptide having amino acid substitutions. Generally,nucleotide sequence variants of the invention will have at least 40%,50%, 60%, to 70%, e.g., 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, to 79%,generally at least 80%, e.g., 81%-84%, at least 85%, e.g., 86%, 87%,88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, to 98%, sequenceidentity to the native (endogenous) nucleotide sequence.

A “transgene” refers to a gene that has been introduced into the genomeby transformation. Transgenes include, for example, DNA that is eitherheterologous or homologous to the DNA of a particular cell to betransformed. Additionally, transgenes may include native genes insertedinto a non-native organism, or chimeric genes.

The term “endogenous gene” refers to a native gene in its naturallocation in the genome of an organism.

The terms “protein,” “peptide” and “polypeptide” are usedinterchangeably herein.

“Wild-type” refers to the normal gene or organism found in nature.

“Genome” refers to the complete genetic material of an organism.

A “vector” is defined to include, inter alia, any viral vector, as wellas any plasmid, cosmid, phage or binary vector in double or singlestranded linear or circular form that may or may not be selftransmissible or mobilisable, and that can transform prokaryotic oreukaryotic host either by integration into the cellular genome or existextrachromosomally (e.g., autonomous replicating plasmid with an originof replication).

“Expression cassette” as used herein means a nucleic acid sequencecapable of directing expression of a particular nucleotide sequence inan appropriate host cell, which may include a promoter operably linkedto the nucleotide sequence of interest that may be operably linked totermination signals. The coding region usually codes for a functionalRNA of interest, for example an siRNA. The expression cassette includingthe nucleotide sequence of interest may be chimeric. The expressioncassette may also be one that is naturally occurring but has beenobtained in a recombinant form useful for heterologous expression. Theexpression of the nucleotide sequence in the expression cassette may beunder the control of a constitutive promoter or of a regulatablepromoter that initiates transcription only when the host cell is exposedto some particular stimulus. In the case of a multicellular organism,the promoter can also be specific to a particular tissue or organ orstage of development.

Such expression cassettes can include a transcriptional initiationregion linked to a nucleotide sequence of interest. Such an expressioncassette is provided with a plurality of restriction sites for insertionof the gene of interest to be under the transcriptional regulation ofthe regulatory regions. The expression cassette may additionally containselectable marker genes.

“Coding sequence” refers to a DNA or RNA sequence that codes for aspecific amino acid sequence. It may constitute an “uninterrupted codingsequence,” i.e., lacking an intron, such as in a cDNA, or it may includeone or more introns bounded by appropriate splice junctions. An “intron”is a sequence of RNA that is contained in the primary transcript but isremoved through cleavage and re-ligation of the RNA within the cell tocreate the mature mRNA that can be translated into a protein.

The term “open reading frame” (ORF) refers to the sequence betweentranslation initiation and termination codons of a coding sequence. Theterms “initiation codon” and “termination codon” refer to a unit ofthree adjacent nucleotides (a ‘codon’) in a coding sequence thatspecifies initiation and chain termination, respectively, of proteinsynthesis (mRNA translation).

“Functional RNA” refers to sense RNA, antisense RNA, ribozyme RNA,siRNA, or other RNA that may not be translated but yet has an effect onat least one cellular process.

The term “RNA transcript” or “transcript” refers to the productresulting from RNA polymerase catalyzed transcription of a DNA sequence.When the RNA transcript is a perfect complementary copy of the DNAsequence, it is referred to as the primary transcript or it may be a RNAsequence derived from posttranscriptional processing of the primarytranscript and is referred to as the mature RNA. “Messenger RNA” (mRNA)refers to the RNA that is without introns and that can be translatedinto protein by the cell.

“cDNA” refers to a single- or a double-stranded DNA that iscomplementary to and derived from mRNA.

“Regulatory sequences” are nucleotide sequences located upstream (5′non-coding sequences), within, or downstream (3′ non-coding sequences)of a coding sequence, and which influence the transcription, RNAprocessing or stability, or translation of the associated codingsequence. Regulatory sequences include enhancers, promoters, translationleader sequences, introns, and polyadenylation signal sequences. Theyinclude natural and synthetic sequences as well as sequences that may bea combination of synthetic and natural sequences. As is noted herein,the term “suitable regulatory sequences” is not limited to promoters.However, some suitable regulatory sequences useful in the presentinvention will include, but are not limited to constitutive promoters,tissue-specific promoters, development-specific promoters, regulatablepromoters and viral promoters.

“5′ non-coding sequence” refers to a nucleotide sequence located 5′(upstream) to the coding sequence. It is present in the fully processedmRNA upstream of the initiation codon and may affect processing of theprimary transcript to mRNA, mRNA stability or translation efficiency.

“3′ non-coding sequence” refers to nucleotide sequences located 3′(downstream) to a coding sequence and may include polyadenylation signalsequences and other sequences encoding regulatory signals capable ofaffecting mRNA processing or gene expression. The polyadenylation signalis usually characterized by affecting the addition of polyadenylic acidtracts to the 3′ end of the mRNA precursor.

The term “translation leader sequence” refers to that DNA sequenceportion of a gene between the promoter and coding sequence that istranscribed into RNA and is present in the fully processed mRNA upstream(5′) of the translation start codon. The translation leader sequence mayaffect processing of the primary transcript to mRNA, mRNA stability ortranslation efficiency.

The term “mature” protein refers to a post-translationally processedpolypeptide without its signal peptide. “Precursor” protein refers tothe primary product of translation of an mRNA. “Signal peptide” refersto the amino terminal extension of a polypeptide, which is translated inconjunction with the polypeptide forming a precursor peptide and whichis required for its entrance into the secretory pathway. The term“signal sequence” refers to a nucleotide sequence that encodes thesignal peptide.

“Promoter” refers to a nucleotide sequence, usually upstream (5′) to itscoding sequence, which directs and/or controls the expression of thecoding sequence by providing the recognition for RNA polymerase andother factors required for proper transcription. “Promoter” includes aminimal promoter that is a short DNA sequence comprised of a TATA-boxand other sequences that serve to specify the site of transcriptioninitiation, to which regulatory elements are added for control ofexpression. “Promoter” also refers to a nucleotide sequence thatincludes a minimal promoter plus regulatory elements that is capable ofcontrolling the expression of a coding sequence or functional RNA. Thistype of promoter sequence consists of proximal and more distal upstreamelements, the latter elements often referred to as enhancers.Accordingly, an “enhancer” is a DNA sequence that can stimulate promoteractivity and may be an innate element of the promoter or a heterologouselement inserted to enhance the level or tissue specificity of apromoter. It is capable of operating in both orientations (normal orflipped), and is capable of functioning even when moved either upstreamor downstream from the promoter. Both enhancers and other upstreampromoter elements bind sequence-specific DNA-binding proteins thatmediate their effects. Promoters may be derived in their entirety from anative gene, or be composed of different elements derived from differentpromoters found in nature, or even be comprised of synthetic DNAsegments. A promoter may also contain DNA sequences that are involved inthe binding of protein factors that control the effectiveness oftranscription initiation in response to physiological or developmentalconditions. Examples of promoters that may be used in the presentinvention include the mouse U6 RNA promoters, synthetic human H1RNApromoters, SV40, CMV, RSV, RNA polymerase II and RNA polymerase IIIpromoters.

The “initiation site” is the position surrounding the first nucleotidethat is part of the transcribed sequence, which is also defined asposition +1. With respect to this site all other sequences of the geneand its controlling regions are numbered. Downstream sequences (i.e.,further protein encoding sequences in the 3′ direction) are denominatedpositive, while upstream sequences (mostly of the controlling regions inthe 5′ direction) are denominated negative.

Promoter elements, particularly a TATA element, that are inactive orthat have greatly reduced promoter activity in the absence of upstreamactivation are referred to as “minimal or core promoters.” In thepresence of a suitable transcription factor, the minimal promoterfunctions to permit transcription. A “minimal or core promoter” thusconsists only of all basal elements needed for transcription initiation,e.g., a TATA box and/or an initiator.

“Constitutive expression” refers to expression using a constitutive orregulated promoter. “Conditional” and “regulated expression” refer toexpression controlled by a regulated promoter.

“Operably-linked” refers to the association of nucleic acid sequences onsingle nucleic acid fragment so that the function of one of thesequences is affected by another. For example, a regulatory DNA sequenceis said to be “operably linked to” or “associated with” a DNA sequencethat codes for an RNA or a polypeptide if the two sequences are situatedsuch that the regulatory DNA sequence affects expression of the codingDNA sequence (i.e., that the coding sequence or functional RNA is underthe transcriptional control of the promoter). Coding sequences can beoperably-linked to regulatory sequences in sense or antisenseorientation.

“Expression” refers to the transcription and/or translation of anendogenous gene, heterologous gene or nucleic acid segment, or atransgene in cells. For example, in the case of siRNA constructs,expression may refer to the transcription of the siRNA only. Inaddition, expression refers to the transcription and stable accumulationof sense (mRNA) or functional RNA. Expression may also refer to theproduction of protein.

“Altered levels” refers to the level of expression in transgenic cellsor organisms that differs from that of normal or untransformed cells ororganisms.

“Overexpression” refers to the level of expression in transgenic cellsor organisms that exceeds levels of expression in normal oruntransformed cells or organisms.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of protein from anendogenous gene or a transgene.

“Transcription stop fragment” refers to nucleotide sequences thatcontain one or more regulatory signals, such as polyadenylation signalsequences, capable of terminating transcription. Examples include the 3′non-regulatory regions of genes encoding nopaline synthase and the smallsubunit of ribulose bisphosphate carboxylase.

“Translation stop fragment” refers to nucleotide sequences that containone or more regulatory signals, such as one or more termination codonsin all three frames, capable of terminating translation. Insertion of atranslation stop fragment adjacent to or near the initiation codon atthe 5′ end of the coding sequence will result in no translation orimproper translation. Excision of the translation stop fragment bysite-specific recombination will leave a site-specific sequence in thecoding sequence that does not interfere with proper translation usingthe initiation codon.

The terms “cis-acting sequence” and “cis-acting element” refer to DNA orRNA sequences whose functions require them to be on the same molecule.An example of a cis-acting sequence on the replicon is the viralreplication origin.

The terms “trans-acting sequence” and “trans-acting element” refer toDNA or RNA sequences whose function does not require them to be on thesame molecule.

“Chromosomally-integrated” refers to the integration of a foreign geneor nucleic acid construct into the host DNA by covalent bonds. Wheregenes are not “chromosomally integrated” they may be “transientlyexpressed.” Transient expression of a gene refers to the expression of agene that is not integrated into the host chromosome but functionsindependently, either as part of an autonomously replicating plasmid orexpression cassette, for example, or as part of another biologicalsystem such as a virus.

The following terms are used to describe the sequence relationshipsbetween two or more nucleic acids or polynucleotides: (a) “referencesequence,” (b) “comparison window,” (c) “sequence identity,” (d)“percentage of sequence identity,” and (e) “substantial identity.”

(a) As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or genesequence.

(b) As used herein, “comparison window” makes reference to a contiguousand specified segment of a polynucleotide sequence, wherein thepolynucleotide sequence in the comparison window may comprise additionsor deletions (i.e., gaps) compared to the reference sequence (which doesnot comprise additions or deletions) for optimal alignment of the twosequences. Generally, the comparison window is at least 20 contiguousnucleotides in length, and optionally can be 30, 40, 50, 100, or longer.Those of skill in the art understand that to avoid a high similarity toa reference sequence due to inclusion of gaps in the polynucleotidesequence a gap penalty is typically introduced and is subtracted fromthe number of matches.

Methods of alignment of sequences for comparison are well-known in theart. Thus, the determination of percent identity between any twosequences can be accomplished using a mathematical algorithm.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the Wisconsin Genetics Software Package, Version 8 (availablefrom Genetics Computer Group (GCG), 575 Science Drive, Madison, Wis.,USA). Alignments using these programs can be performed using the defaultparameters.

Software for performing BLAST analyses is publicly available through theNational Center for Biotechnology Information. This algorithm involvesfirst identifying high scoring sequence pairs (HSPs) by identifyingshort words of length W in the query sequence, which either match orsatisfy some positive-valued threshold score T when aligned with a wordof the same length in a database sequence. T is referred to as theneighborhood word score threshold. These initial neighborhood word hitsact as seeds for initiating searches to find longer HSPs containingthem. The word hits are then extended in both directions along eachsequence for as far as the cumulative alignment score can be increased.Cumulative scores are calculated using, for nucleotide sequences, theparameters M (reward score for a pair of matching residues; always>0)and N (penalty score for mismatching residues; always<0). For amino acidsequences, a scoring matrix is used to calculate the cumulative score.Extension of the word hits in each direction are halted when thecumulative alignment score falls off by the quantity X from its maximumachieved value, the cumulative score goes to zero or below due to theaccumulation of one or more negative-scoring residue alignments, or theend of either sequence is reached.

In addition to calculating percent sequence identity, the BLASTalgorithm also performs a statistical analysis of the similarity betweentwo sequences. One measure of similarity provided by the BLAST algorithmis the smallest sum probability (P(N)), which provides an indication ofthe probability by which a match between two nucleotide sequences wouldoccur by chance. For example, a test nucleic acid sequence is consideredsimilar to a reference sequence if the smallest sum probability in acomparison of the test nucleic acid sequence to the reference nucleicacid sequence is less than about 0.1, more preferably less than about0.01, and most preferably less than about 0.001.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. When utilizing BLAST, Gapped BLAST, PSI-BLAST, thedefault parameters of the respective programs (e.g. BLASTN fornucleotide sequences) can be used. The BLASTN program (for nucleotidesequences) uses as defaults a wordlength (W) of 11, an expectation (E)of 10, a cutoff of 100, M=5, N=−4, and a comparison of both strands.Alignment may also be performed manually by inspection.

For purposes of the present invention, comparison of nucleotidesequences for determination of percent sequence identity to the promotersequences disclosed herein is preferably made using the BlastN program(version 1.4.7 or later) with its default parameters or any equivalentprogram. By “equivalent program” is intended any sequence comparisonprogram that, for any two sequences in question, generates an alignmenthaving identical nucleotide matches and an identical percent sequenceidentity when compared to the corresponding alignment generated by thepreferred program.

(c) As used herein, “sequence identity” or “identity” in the context oftwo nucleic acid sequences makes reference to a specified percentage ofnucleotides in the two sequences that are the same when aligned formaximum correspondence over a specified comparison window, as measuredby sequence comparison algorithms or by visual inspection.

(d) As used herein, “percentage of sequence identity” means the valuedetermined by comparing two optimally aligned sequences over acomparison window, wherein the portion of the polynucleotide sequence inthe comparison window may comprise additions or deletions (i.e., gaps)as compared to the reference sequence (which does not comprise additionsor deletions) for optimal alignment of the two sequences. The percentageis calculated by determining the number of positions at which theidentical nucleic acid base or amino acid residue occurs in bothsequences to yield the number of matched positions, dividing the numberof matched positions by the total number of positions in the window ofcomparison, and multiplying the result by 100 to yield the percentage ofsequence identity.

(e) The term “substantial identity” of polynucleotide sequences meansthat a polynucleotide comprises a sequence that has at least 70%, 71%,72%, 73%, 74%, 75%, 76%, 77%, 78%, or 79%, preferably at least 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, or 89%, more preferably at least 90%,91%, 92%, 93%, or 94%, and most preferably at least 95%, 96%, 97%, 98%,or 99% sequence identity, compared to a reference sequence using one ofthe alignment programs described using standard parameters.

Another indication that nucleotide sequences are substantially identicalis if two molecules hybridize to each other under stringent conditions.Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence at adefined ionic strength and pH. However, stringent conditions encompasstemperatures in the range of about 1° C. to about 20° C., depending uponthe desired degree of stringency as otherwise qualified herein.

For sequence comparison, typically one sequence acts as a referencesequence to which test sequences are compared. When using a sequencecomparison algorithm, test and reference sequences are input into acomputer, subsequence coordinates are designated if necessary, andsequence algorithm program parameters are designated. The sequencecomparison algorithm then calculates the percent sequence identity forthe test sequence(s) relative to the reference sequence, based on thedesignated program parameters.

As noted herein, another indication that two nucleic acid sequences aresubstantially identical is that the two molecules hybridize to eachother under stringent conditions. The phrase “hybridizing specificallyto” refers to the binding, duplexing, or hybridizing of a molecule onlyto a particular nucleotide sequence under stringent conditions when thatsequence is present in a complex mixture (e.g., total cellular) DNA orRNA. “Bind(s) substantially” refers to complementary hybridizationbetween a probe nucleic acid and a target nucleic acid and embracesminor mismatches that can be accommodated by reducing the stringency ofthe hybridization media to achieve the desired detection of the targetnucleic acid sequence.

“Stringent hybridization conditions” and “stringent hybridization washconditions” in the context of nucleic acid hybridization experimentssuch as Southern and Northern hybridizations are sequence dependent, andare different under different environmental parameters. Longer sequenceshybridize specifically at higher temperatures. The Tm is the temperature(under defined ionic strength and pH) at which 50% of the targetsequence hybridizes to a perfectly matched probe. Specificity istypically the function of post-hybridization washes, the criticalfactors being the ionic strength and temperature of the final washsolution. For DNA-DNA hybrids, the Tm can be approximated from theequation: Tm 81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L;where M is the molarity of monovalent cations, % GC is the percentage ofguanosine and cytosine nucleotides in the DNA, % form is the percentageof formamide in the hybridization solution, and L is the length of thehybrid in base pairs. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with >90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9, or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15, or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired T, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a T of less than 45° C. (aqueous solution) or 32°C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. Generally,highly stringent hybridization and wash conditions are selected to beabout 5° C. lower than the thermal melting point (Tm) for the specificsequence at a defined ionic strength and pH.

An example of highly stringent wash conditions is 0.15 M NaCl at 72° C.for about 15 minutes. An example of stringent wash conditions is a0.2×SSC wash at 65° C. for 15 minutes (see, Sambrook and Russell 2001,for a description of SSC buffer). Often, a high stringency wash ispreceded by a low stringency wash to remove background probe signal. Forshort nucleic acid sequences (e.g., about 10 to 50 nucleotides),stringent conditions typically involve salt concentrations of less thanabout 1.5 M, more preferably about 0.01 to 1.0 M, Na ion concentration(or other salts) at pH 7.0 to 8.3, and the temperature is typically atleast about 30° C. Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. In general, a signalto noise ratio of 2× (or higher) than that observed for an unrelatedprobe in the particular hybridization assay indicates detection of aspecific hybridization. Very stringent conditions are selected to beequal to the Tm for a particular nucleic acid molecule.

Very stringent conditions are selected to be equal to the T_(m) for aparticular probe. An example of stringent conditions for hybridizationof complementary nucleic acids which have more than 100 complementaryresidues on a filter in a Southern or Northern blot is 50% formamide,e.g., hybridization in 50% formamide, 1 M NaCl, 1% SDS at 37° C., and awash in 0.1×SSC at 60 to 65° C. Exemplary low stringency conditionsinclude hybridization with a buffer solution of 30 to 35% formamide, 1MNaCl, 1% SDS (sodium dodecyl sulfate) at 37° C., and a wash in 1× to2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at 50 to 55° C.Exemplary moderate stringency conditions include hybridization in 40 to45% formamide, 1.0 M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSCat 55 to 60° C.

The term “transformation” refers to the transfer of a nucleic acidfragment into the genome of a host cell, resulting in genetically stableinheritance. A “host cell” is a cell that has been transformed, or iscapable of transformation, by an exogenous nucleic acid molecule. Hostcells containing the transformed nucleic acid fragments are referred toas “transgenic” cells.

“Transformed,” “transduced,” “transgenic” and “recombinant” refer to ahost cell into which a heterologous nucleic acid molecule has beenintroduced. As used herein the term “transfection” refers to thedelivery of DNA into eukaryotic (e.g., mammalian) cells. The term“transformation” is used herein to refer to delivery of DNA intoprokaryotic (e.g., E. coli) cells. The term “transduction” is usedherein to refer to infecting cells with viral particles. The nucleicacid molecule can be stably integrated into the genome generally knownin the art. Known methods of PCR include, but are not limited to,methods using paired primers, nested primers, single specific primers,degenerate primers, gene-specific primers, vector-specific primers,partially mismatched primers, and the like. For example, “transformed,”“transformant,” and “transgenic” cells have been through thetransformation process and contain a foreign gene integrated into theirchromosome. The term “untransformed” refers to normal cells that havenot been through the transformation process.

“Genetically altered cells” denotes cells which have been modified bythe introduction of recombinant or heterologous nucleic acids (e.g., oneor more DNA constructs or their RNA counterparts) and further includesthe progeny of such cells which retain part or all of such geneticmodification.

As used herein, the term “derived” or “directed to” with respect to anucleotide molecule means that the molecule has complementary sequenceidentity to a particular molecule of interest.

The siRNAs of the present invention can be generated by any method knownto the art, for example, by in vitro transcription, recombinantly, or bysynthetic means. In one example, the siRNAs can be generated in vitro byusing a recombinant enzyme, such as T7 RNA polymerase, and DNAoligonucleotide templates.

Nucleic Acid Molecules of the Invention

The terms “isolated and/or purified” refer to in vitro isolation of anucleic acid, e.g., a DNA or RNA molecule from its natural cellularenvironment, and from association with other components of the cell,such as nucleic acid or polypeptide, so that it can be sequenced,replicated, and/or expressed. The RNA or DNA is “isolated” in that it isfree from at least one contaminating nucleic acid with which it isnormally associated in the natural source of the RNA or DNA and ispreferably substantially free of any other mammalian RNA or DNA. Thephrase “free from at least one contaminating source nucleic acid withwhich it is normally associated” includes the case where the nucleicacid is reintroduced into the source or natural cell but is in adifferent chromosomal location or is otherwise flanked by nucleic acidsequences not normally found in the source cell, e.g., in a vector orplasmid.

In addition to a DNA sequence encoding a siRNA, the nucleic acidmolecules of the invention include double-stranded interfering RNAmolecules, which are also useful to inhibit expression of a target gene.

As used herein, the term “recombinant nucleic acid”, e.g., “recombinantDNA sequence or segment” refers to a nucleic acid, e.g., to DNA, thathas been derived or isolated from any appropriate cellular source, thatmay be subsequently chemically altered in vitro, so that its sequence isnot naturally occurring, or corresponds to naturally occurring sequencesthat are not positioned as they would be positioned in a genome whichhas not been transformed with exogenous DNA. An example of preselectedDNA “derived” from a source would be a DNA sequence that is identifiedas a useful fragment within a given organism, and which is thenchemically synthesized in essentially pure form. An example of such DNA“isolated” from a source would be a useful DNA sequence that is excisedor removed from a source by chemical means, e.g., by the use ofrestriction endonucleases, so that it can be further manipulated, e.g.,amplified, for use in the invention, by the methodology of geneticengineering. “Recombinant DNA” includes completely synthetic DNAsequences, semi-synthetic DNA sequences, DNA sequences isolated frombiological sources, and DNA sequences derived from RNA, as well asmixtures thereof.

Expression Cassettes of the Invention

To prepare expression cassettes, the recombinant DNA sequence or segmentmay be circular or linear, double-stranded or single-stranded.Generally, the DNA sequence or segment is in the form of chimeric DNA,such as plasmid DNA or a vector that can also contain coding regionsflanked by control sequences that promote the expression of therecombinant DNA present in the resultant transformed cell.

A “chimeric” vector or expression cassette, as used herein, means avector or cassette including nucleic acid sequences from at least twodifferent species, or has a nucleic acid sequence from the same speciesthat is linked or associated in a manner that does not occur in the“native” or wild type of the species.

Aside from recombinant DNA sequences that serve as transcription unitsfor an RNA transcript, or portions thereof, a portion of the recombinantDNA may be untranscribed, serving a regulatory or a structural function.For example, the recombinant DNA may have a promoter that is active inmammalian cells.

Other elements functional in the host cells, such as introns, enhancers,polyadenylation sequences and the like, may also be a part of therecombinant DNA. Such elements may or may not be necessary for thefunction of the DNA, but may provide improved expression of the DNA byaffecting transcription, stability of the siRNA, or the like. Suchelements may be included in the DNA as desired to obtain the optimalperformance of the siRNA in the cell.

Control sequences are DNA sequences necessary for the expression of anoperably linked coding sequence in a particular host organism. Thecontrol sequences that are suitable for prokaryotic cells, for example,include a promoter, and optionally an operator sequence, and a ribosomebinding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

Operably linked nucleic acids are nucleic acids placed in a functionalrelationship with another nucleic acid sequence. For example, a promoteror enhancer is operably linked to a coding sequence if it affects thetranscription of the sequence; or a ribosome binding site is operablylinked to a coding sequence if it is positioned so as to facilitatetranslation. Generally, operably linked DNA sequences are DNA sequencesthat are linked are contiguous. However, enhancers do not have to becontiguous. Linking is accomplished by ligation at convenientrestriction sites. If such sites do not exist, the syntheticoligonucleotide adaptors or linkers are used in accord with conventionalpractice.

The recombinant DNA to be introduced into the cells may contain either aselectable marker gene or a reporter gene or both to facilitateidentification and selection of expressing cells from the population ofcells sought to be transfected or infected through viral vectors. Inother embodiments, the selectable marker may be carried on a separatepiece of DNA and used in a co-transfection procedure. Both selectablemarkers and reporter genes may be flanked with appropriate regulatorysequences to enable expression in the host cells. Useful selectablemarkers are known in the art and include, for example,antibiotic-resistance genes, such as neo and the like.

Reporter genes are used for identifying potentially transfected cellsand for evaluating the functionality of regulatory sequences. Reportergenes that encode for easily assayable proteins are well known in theart. In general, a reporter gene is a gene that is not present in orexpressed by the recipient organism or tissue and that encodes a proteinwhose expression is manifested by some easily detectable property, e.g.,enzymatic activity. For example, reporter genes include thechloramphenicol acetyl transferase gene (cat) from Tn9 of E. coli andthe luciferase gene from firefly Photinus pyralis. Expression of thereporter gene is assayed at a suitable time after the DNA has beenintroduced into the recipient cells.

The general methods for constructing recombinant DNA that can transfecttarget cells are well known to those skilled in the art, and the samecompositions and methods of construction may be utilized to produce theDNA useful herein.

The recombinant DNA can be readily introduced into the host cells, e.g.,mammalian, bacterial, yeast or insect cells by transfection with anexpression vector composed of DNA encoding the siRNA by any procedureuseful for the introduction into a particular cell, e.g., physical orbiological methods, to yield a cell having the recombinant DNA stablyintegrated into its genome or existing as a episomal element, so thatthe DNA molecules, or sequences of the present invention are expressedby the host cell. Preferably, the DNA is introduced into host cells viaa vector. The host cell is preferably of eukaryotic origin, e.g., plant,mammalian, insect, yeast or fungal sources, but host cells ofnon-eukaryotic origin may also be employed.

Physical methods to introduce a preselected DNA into a host cell includecalcium phosphate precipitation, lipofection, particle bombardment,microinjection, electroporation, and the like. Biological methods tointroduce the DNA of interest into a host cell include the use of DNAand RNA viral vectors. For mammalian gene therapy, as described hereinbelow, it is desirable to use an efficient means of inserting a copygene into the host genome. Viral vectors, and especially retroviralvectors, have become the most widely used method for inserting genesinto mammalian, e.g., human cells. Other viral vectors can be derivedfrom poxviruses, herpes simplex virus I, adenoviruses andadeno-associated viruses, and the like. See, for example, U.S. Pat. Nos.5,350,674 and 5,585,362.

As discussed herein, a “transfected” “or “transduced” host cell or cellline is one in which the genome has been altered or augmented by thepresence of at least one heterologous or recombinant nucleic acidsequence. The host cells of the present invention are typically producedby transfection with a DNA sequence in a plasmid expression vector, aviral expression vector, or as an isolated linear DNA sequence. Thetransfected DNA can become a chromosomally integrated recombinant DNAsequence, which is composed of sequence encoding the siRNA.

To confirm the presence of the recombinant DNA sequence in the hostcell, a variety of assays may be performed. Such assays include, forexample, “molecular biological” assays well known to those of skill inthe art, such as Southern and Northern blotting, RT-PCR and PCR;“biochemical” assays, such as detecting the presence or absence of aparticular peptide, e.g., by immunological means (ELISAs and Westernblots) or by assays described herein to identify agents falling withinthe scope of the invention.

To detect and quantitate RNA produced from introduced recombinant DNAsegments, RT-PCR may be employed. In this application of PCR, it isfirst necessary to reverse transcribe RNA into DNA, using enzymes suchas reverse transcriptase, and then through the use of conventional PCRtechniques amplify the DNA. In most instances PCR techniques, whileuseful, will not demonstrate integrity of the RNA product. Furtherinformation about the nature of the RNA product may be obtained byNorthern blotting. This technique demonstrates the presence of an RNAspecies and gives information about the integrity of that RNA. Thepresence or absence of an RNA species can also be determined using dotor slot blot Northern hybridizations. These techniques are modificationsof Northern blotting and only demonstrate the presence or absence of anRNA species.

While Southern blotting and PCR may be used to detect the recombinantDNA segment in question, they do not provide information as to whetherthe preselected DNA segment is being expressed. Expression may beevaluated by specifically identifying the peptide products of theintroduced recombinant DNA sequences or evaluating the phenotypicchanges brought about by the expression of the introduced recombinantDNA segment in the host cell.

The instant invention provides a cell expression system for expressingexogenous nucleic acid material in a mammalian recipient. The expressionsystem, also referred to as a “genetically modified cell,” comprises acell and an expression vector for expressing the exogenous nucleic acidmaterial. The genetically modified cells are suitable for administrationto a mammalian recipient, where they replace the endogenous cells of therecipient. Thus, the preferred genetically modified cells arenon-immortalized and are non-tumorigenic.

According to one embodiment, the cells are transfected or otherwisegenetically modified ex vivo. The cells are isolated from a mammal(preferably a human), nucleic acid introduced (i.e., transduced ortransfected in vitro) with a vector for expressing a heterologous (e.g.,recombinant) gene encoding the therapeutic agent, and then administeredto a mammalian recipient for delivery of the therapeutic agent in situ.The mammalian recipient may be a human and the cells to be modified areautologous cells, i.e., the cells are isolated from the mammalianrecipient.

According to another embodiment, the cells are transfected or transducedor otherwise genetically modified in vivo. The cells from the mammalianrecipient are transduced or transfected in vivo with a vector containingexogenous nucleic acid material for expressing a heterologous (e.g.,recombinant) gene encoding a therapeutic agent and the therapeutic agentis delivered in situ.

As used herein, “exogenous nucleic acid material” refers to a nucleicacid or an oligonucleotide, either natural or synthetic, which is notnaturally found in the cells; or if it is naturally found in the cells,is modified from its original or native form. Thus, “exogenous nucleicacid material” includes, for example, a non-naturally occurring nucleicacid that can be transcribed into an anti-sense RNA, a siRNA, as well asa “heterologous gene” (i.e., a gene encoding a protein that is notexpressed or is expressed at biologically insignificant levels in anaturally-occurring cell of the same type). To illustrate, a syntheticor natural gene encoding human erythropoietin (EPO) would be considered“exogenous nucleic acid material” with respect to human peritonealmesothelial cells since the latter cells do not naturally express EPO.Still another example of “exogenous nucleic acid material” is theintroduction of only part of a gene to create a recombinant gene, suchas combining an regulatable promoter with an endogenous coding sequencevia homologous recombination.

The condition amenable to gene inhibition therapy may be a prophylacticprocess, i.e., a process for preventing disease or an undesired medicalcondition. Thus, the instant invention embraces a system for deliveringsiRNA that has a prophylactic function (i.e., a prophylactic agent) tothe mammalian recipient.

Methods for Introducing the Expression Cassettes of the Invention intoCells

The inhibitory nucleic acid material (e.g., an expression cassetteencoding siRNA directed to a gene of interest) can be introduced intothe cell ex vivo or in vivo by genetic transfer methods, such astransfection or transduction, to provide a genetically modified cell.Various expression vectors (i.e., vehicles for facilitating delivery ofexogenous nucleic acid into a target cell) are known to one of ordinaryskill in the art.

As used herein, “transfection of cells” refers to the acquisition by acell of new nucleic acid material by incorporation of added DNA. Thus,transfection refers to the insertion of nucleic acid into a cell usingphysical or chemical methods. Several transfection techniques are knownto those of ordinary skill in the art including calcium phosphate DNAco-precipitation, DEAE-dextran, electroporation, cationicliposome-mediated transfection, tungsten particle-facilitatedmicroparticle bombardment, and strontium phosphate DNA co-precipitation.

In contrast, “transduction of cells” refers to the process oftransferring nucleic acid into a cell using a DNA or RNA virus. A RNAvirus (i.e., a retrovirus) for transferring a nucleic acid into a cellis referred to herein as a transducing chimeric retrovirus. Exogenousnucleic acid material contained within the retrovirus is incorporatedinto the genome of the transduced cell. A cell that has been transducedwith a chimeric DNA virus (e.g., an adenovirus carrying a cDNA encodinga therapeutic agent), will not have the exogenous nucleic acid materialincorporated into its genome but will be capable of expressing theexogenous nucleic acid material that is retained extrachromosomallywithin the cell.

The exogenous nucleic acid material can include the nucleic acidencoding the siRNA together with a promoter to control transcription.The promoter characteristically has a specific nucleotide sequencenecessary to initiate transcription. The exogenous nucleic acid materialmay further include additional sequences (i.e., enhancers) required toobtain the desired gene transcription activity. For the purpose of thisdiscussion an “enhancer” is simply any non-translated DNA sequence thatworks with the coding sequence (in cis) to change the basaltranscription level dictated by the promoter. The exogenous nucleic acidmaterial may be introduced into the cell genome immediately downstreamfrom the promoter so that the promoter and coding sequence areoperatively linked so as to permit transcription of the coding sequence.An expression vector can include an exogenous promoter element tocontrol transcription of the inserted exogenous gene. Such exogenouspromoters include both constitutive and regulatable promoters.

Naturally-occurring constitutive promoters control the expression ofessential cell functions. As a result, a nucleic acid sequence under thecontrol of a constitutive promoter is expressed under all conditions ofcell growth. Constitutive promoters include the promoters for thefollowing genes which encode certain constitutive or “housekeeping”functions: hypoxanthine phosphoribosyl transferase (HPRT), dihydrofolatereductase (DHFR), adenosine deaminase, phosphoglycerol kinase (PGK),pyruvate kinase, phosphoglycerol mutase, the beta-actin promoter, andother constitutive promoters known to those of skill in the art. Inaddition, many viral promoters function constitutively in eukaryoticcells. These include: the early and late promoters of SV40; the longterminal repeats (LTRs) of Moloney Leukemia Virus and otherretroviruses; and the thymidine kinase promoter of Herpes Simplex Virus,among many others.

Nucleic acid sequences that are under the control of regulatablepromoters are expressed only or to a greater or lesser degree in thepresence of an inducing or repressing agent, (e.g., transcription undercontrol of the metallothionein promoter is greatly increased in presenceof certain metal ions). Regulatable promoters include responsiveelements (REs) that stimulate transcription when their inducing factorsare bound. For example, there are REs for serum factors, steroidhormones, retinoic acid, cyclic AMP, and tetracycline and doxycycline.Promoters containing a particular RE can be chosen in order to obtain anregulatable response and in some cases, the RE itself may be attached toa different promoter, thereby conferring regulatability to the encodednucleic acid sequence. Thus, by selecting the appropriate promoter(constitutive versus regulatable; strong versus weak), it is possible tocontrol both the existence and level of expression of a nucleic acidsequence in the genetically modified cell. If the nucleic acid sequenceis under the control of an regulatable promoter, delivery of thetherapeutic agent in situ is triggered by exposing the geneticallymodified cell in situ to conditions for permitting transcription of thenucleic acid sequence, e.g., by intraperitoneal injection of specificinducers of the regulatable promoters which control transcription of theagent. For example, in situ expression of a nucleic acid sequence underthe control of the metallothionein promoter in genetically modifiedcells is enhanced by contacting the genetically modified cells with asolution containing the appropriate (i.e., inducing) metal ions in situ.

Accordingly, the amount of siRNA generated in situ is regulated bycontrolling such factors as the nature of the promoter used to directtranscription of the nucleic acid sequence, (i.e., whether the promoteris constitutive or regulatable, strong or weak) and the number of copiesof the exogenous nucleic acid sequence encoding a siRNA sequence thatare in the cell.

In addition to at least one promoter and at least one heterologousnucleic acid sequence encoding the siRNA, the expression vector mayinclude a selection gene, for example, a neomycin resistance gene, forfacilitating selection of cells that have been transfected or transducedwith the expression vector.

Cells can also be transfected with two or more expression vectors, atleast one vector containing the nucleic acid sequence(s) encoding thesiRNA(s), the other vector containing a selection gene. The selection ofa suitable promoter, enhancer, selection gene, and/or signal sequence isdeemed to be within the scope of one of ordinary skill in the artwithout undue experimentation.

The following discussion is directed to various utilities of the instantinvention. For example, the instant invention has utility as anexpression system suitable for silencing the expression of gene(s) ofinterest.

The instant invention also provides methods for genetically modifyingcells of a mammalian recipient in vivo. According to one embodiment, themethod comprises introducing an expression vector for expressing a siRNAsequence in cells of the mammalian recipient in situ by, for example,injecting the vector into the recipient.

Delivery Vehicles for the Expression Cassettes of the Invention

Delivery of compounds into tissues and across the blood-brain barriercan be limited by the size and biochemical properties of the compounds.Currently, efficient delivery of compounds into cells in vivo can beachieved only when the molecules are small (usually less than 600Daltons). Gene transfer for the correction of inborn errors ofmetabolism and neurodegenerative diseases of the central nervous system(CNS), and for the treatment of cancer has been accomplished withrecombinant adenoviral vectors.

The selection and optimization of a particular expression vector forexpressing a specific siRNA in a cell can be accomplished by obtainingthe nucleic acid sequence of the siRNA, possibly with one or moreappropriate control regions (e.g., promoter, insertion sequence);preparing a vector construct comprising the vector into which isinserted the nucleic acid sequence encoding the siRNA; transfecting ortransducing cultured cells in vitro with the vector construct; anddetermining whether the siRNA is present in the cultured cells.

Vectors for cell gene therapy include viruses, such asreplication-deficient viruses (described in detail below). Exemplaryviral vectors are derived from Harvey Sarcoma virus, ROUS Sarcoma virus,(MPSV), Moloney murine leukemia virus and DNA viruses (e.g.,adenovirus).

Replication-deficient retroviruses are capable of directing synthesis ofall virion proteins, but are incapable of making infectious particles.Accordingly, these genetically altered retroviral expression vectorshave general utility for high-efficiency transduction of nucleic acidsequences in cultured cells, and specific utility for use in the methodof the present invention. Such retroviruses further have utility for theefficient transduction of nucleic acid sequences into cells in vivo.Retroviruses have been used extensively for transferring nucleic acidmaterial into cells. Protocols for producing replication-deficientretroviruses (including the steps of incorporation of exogenous nucleicacid material into a plasmid, transfection of a packaging cell line withplasmid, production of recombinant retroviruses by the packaging cellline, collection of viral particles from tissue culture media, andinfection of the target cells with the viral particles) are well knownin the art.

An advantage of using retroviruses for gene therapy is that the virusesinsert the nucleic acid sequence encoding the siRNA into the host cellgenome, thereby permitting the nucleic acid sequence encoding the siRNAto be passed on to the progeny of the cell when it divides. Promotersequences in the LTR region have can enhance expression of an insertedcoding sequence in a variety of cell types. Some disadvantages of usinga retrovirus expression vector are (1) insertional mutagenesis, i.e.,the insertion of the nucleic acid sequence encoding the siRNA into anundesirable position in the target cell genome which, for example, leadsto unregulated cell growth and (2) the need for target cellproliferation in order for the nucleic acid sequence encoding the siRNAcarried by the vector to be integrated into the target genome.

Another viral candidate useful as an expression vector fortransformation of cells is the adenovirus, a double-stranded DNA virus.The adenovirus is infective in a wide range of cell types, including,for example, muscle and endothelial cells.

Adenoviruses (Ad) are double-stranded linear DNA viruses with a 36 kbgenome. Several features of adenovirus have made them useful astransgene delivery vehicles for therapeutic applications, such asfacilitating in vivo gene delivery. Recombinant adenovirus vectors havebeen shown to be capable of efficient in situ gene transfer toparenchymal cells of various organs, including the lung, brain,pancreas, gallbladder, and liver. This has allowed the use of thesevectors in methods for treating inherited genetic diseases, such ascystic fibrosis, where vectors may be delivered to a target organ. Inaddition, the ability of the adenovirus vector to accomplish in situtumor transduction has allowed the development of a variety ofanticancer gene therapy methods for non-disseminated disease. In thesemethods, vector containment favors tumor cell-specific transduction.

Like the retrovirus, the adenovirus genome is adaptable for use as anexpression vector for gene therapy, i.e., by removing the geneticinformation that controls production of the virus itself. Because theadenovirus functions in an extrachromosomal fashion, the recombinantadenovirus does not have the theoretical problem of insertionalmutagenesis.

Several approaches traditionally have been used to generate therecombinant adenoviruses. One approach involves direct ligation ofrestriction endonuclease fragments containing a nucleic acid sequence ofinterest to portions of the adenoviral genome. Alternatively, thenucleic acid sequence of interest may be inserted into a defectiveadenovirus by homologous recombination results. The desired recombinantsare identified by screening individual plaques generated in a lawn ofcomplementation cells.

Most adenovirus vectors are based on the adenovirus type 5 (Ad5)backbone in which an expression cassette containing the nucleic acidsequence of interest has been introduced in place of the early region 1(E1) or early region 3 (E3). Viruses in which E1 has been deleted aredefective for replication and are propagated in human complementationcells (e.g., 293 or 911 cells), which supply the missing gene E1 and pIXin trans.

In one embodiment of the present invention, one will desire to generatesiRNA in a brain cell or brain tissue. A suitable vector for thisapplication is an FIV vector or an AAV vector. For example, one may useAAV5. Also, one may apply poliovirus or HSV vectors.

Application of siRNA is generally accomplished by transfection ofsynthetic siRNAs, in vitro synthesized RNAs, or plasmids expressingshRNAs or miRNAs. More recently, viruses have been employed for in vitrostudies and to generate transgenic mouse knock-downs of targeted genes.Recombinant adenovirus, adeno-associated virus (AAV) and felineimmunodeficiency virus (FIV) can be used to deliver genes in vitro andin vivo. Each has its own advantages and disadvantages. Adenoviruses aredouble stranded DNA viruses with large genomes (36 kb) and have beenengineered by my laboratory and others to accommodate expressioncassettes in distinct regions.

Adeno-associated viruses have encapsidated genomes, similar to Ad, butare smaller in size and packaging capacity (˜30 nm vs. ˜100 nm;packaging limit of ˜4.5 kb). AAV contain single stranded DNA genomes ofthe + or the − strand. Eight serotypes of AAV (1-8) have been studiedextensively, three of which have been evaluated in the brain. Animportant consideration for the present application is that AAV5transduces striatal and cortical neurons, and is not associated with anyknown pathologies.

Adeno associated virus (AAV) is a small nonpathogenic virus of theparvoviridae family. AAV is distinct from the other members of thisfamily by its dependence upon a helper virus for replication. In theabsence of a helper virus, AAV may integrate in a locus specific mannerinto the q-arm of chromosome 19. The approximately 5 kb genome of AAVconsists of one segment of single stranded DNA of either plus or minuspolarity. The ends of the genome are short inverted terminal repeatswhich can fold into hairpin structures and serve as the origin of viralDNA replication. Physically, the parvovirus virion is non-enveloped andits icosohedral capsid is approximately 20 nm in diameter.

Further provided by this invention are chimeric viruses where AAV can becombined with herpes virus, herpes virus amplicons, baculovirus or otherviruses to achieve a desired tropism associated with another virus. Forexample, the AAV4 ITRs could be inserted in the herpes virus and cellscould be infected. Post-infection, the ITRs of AAV4 could be acted on byAAV4 rep provided in the system or in a separate vehicle to rescue AAV4from the genome. Therefore, the cellular tropism of the herpes simplexvirus can be combined with AAV4 rep mediated targeted integration. Otherviruses that could be utilized to construct chimeric viruses includelentivirus, retrovirus, pseudotyped retroviral vectors, and adenoviralvectors.

Also provided by this invention are variant AAV vectors. For example,the sequence of a native AAV, such as AAV5, can be modified atindividual nucleotides. The present invention includes native and mutantAAV vectors. The present invention further includes all AAV serotypes.

FIV is an enveloped virus with a strong safety profile in humans;individuals bitten or scratched by FIV-infected cats do not seroconvertand have not been reported to show any signs of disease. Like AAV, FIVprovides lasting transgene expression in mouse and nonhuman primateneurons, and transduction can be directed to different cell types bypseudotyping, the process of exchanging the virus's native envelope foran envelope from another virus.

Thus, as will be apparent to one of ordinary skill in the art, a varietyof suitable viral expression vectors are available for transferringexogenous nucleic acid material into cells. The selection of anappropriate expression vector to express a therapeutic agent for aparticular condition amenable to gene silencing therapy and theoptimization of the conditions for insertion of the selected expressionvector into the cell, are within the scope of one of ordinary skill inthe art without the need for undue experimentation.

In another embodiment, the expression vector is in the form of aplasmid, which is transferred into the target cells by one of a varietyof methods: physical (e.g., microinjection, electroporation, scrapeloading, microparticle bombardment) or by cellular uptake as a chemicalcomplex (e.g., calcium or strontium co-precipitation, complexation withlipid, complexation with ligand). Several commercial products areavailable for cationic liposome complexation including Lipofectin™(Gibco-BRL, Gaithersburg, Md.) and Transfectam™ (Promega®, Madison,Wis.). However, the efficiency of transfection by these methods ishighly dependent on the nature of the target cell and accordingly, theconditions for optimal transfection of nucleic acids into cells usingthe herein-mentioned procedures must be optimized. Such optimization iswithin the scope of one of ordinary skill in the art without the needfor undue experimentation.

Dosages, Formulations and Routes of Administration of the Agents of theInvention

The agents of the invention are preferably administered so as to resultin a reduction in at least one symptom associated with a disease. Theamount administered will vary depending on various factors including,but not limited to, the composition chosen, the particular disease, theweight, the physical condition, and the age of the mammal, and whetherprevention or treatment is to be achieved. Such factors can be readilydetermined by the clinician employing animal models or other testsystems, which are well known to the art. As used herein, the term“therapeutic siRNA” refers to any siRNA that has a beneficial effect onthe recipient. Thus, “therapeutic siRNA” embraces both therapeutic andprophylactic siRNA.

Administration of siRNA may be accomplished through the administrationof the nucleic acid molecule encoding the siRNA. Pharmaceuticalformulations, dosages and routes of administration for nucleic acids aregenerally known.

The present invention envisions treating Huntington's disease in amammal by the administration of an agent, e.g., a nucleic acidcomposition, an expression vector, or a viral particle of the invention.Administration of the therapeutic agents in accordance with the presentinvention may be continuous or intermittent, depending, for example,upon the recipient's physiological condition, whether the purpose of theadministration is therapeutic or prophylactic, and other factors knownto skilled practitioners. The administration of the agents of theinvention may be essentially continuous over a preselected period oftime or may be in a series of spaced doses. Both local and systemicadministration is contemplated.

One or more suitable unit dosage forms having the therapeutic agent(s)of the invention, which, as discussed below, may optionally beformulated for sustained release (for example using microencapsulation,see WO 94/07529, and U.S. Pat. No. 4,962,091 the disclosures of whichare incorporated by reference herein), can be administered by a varietyof routes including parenteral, including by intravenous andintramuscular routes, as well as by direct injection into the diseasedtissue. For example, the therapeutic agent may be directly injected intothe brain. Alternatively the therapeutic agent may be introducedintrathecally for brain and spinal cord conditions. In another example,the therapeutic agent may be introduced intramuscularly for viruses thattraffic back to affected neurons from muscle, such as AAV, lentivirusand adenovirus. The formulations may, where appropriate, be convenientlypresented in discrete unit dosage forms and may be prepared by any ofthe methods well known to pharmacy. Such methods may include the step ofbringing into association the therapeutic agent with liquid carriers,solid matrices, semi-solid carriers, finely divided solid carriers orcombinations thereof, and then, if necessary, introducing or shaping theproduct into the desired delivery system.

When the therapeutic agents of the invention are prepared foradministration, they are preferably combined with a pharmaceuticallyacceptable carrier, diluent or excipient to form a pharmaceuticalformulation, or unit dosage form. The total active ingredients in suchformulations include from 0.1 to 99.9% by weight of the formulation. A“pharmaceutically acceptable” is a carrier, diluent, excipient, and/orsalt that is compatible with the other ingredients of the formulation,and not deleterious to the recipient thereof. The active ingredient foradministration may be present as a powder or as granules, as a solution,a suspension or an emulsion.

Pharmaceutical formulations containing the therapeutic agents of theinvention can be prepared by procedures known in the art using wellknown and readily available ingredients. The therapeutic agents of theinvention can also be formulated as solutions appropriate for parenteraladministration, for instance by intramuscular, subcutaneous orintravenous routes.

The pharmaceutical formulations of the therapeutic agents of theinvention can also take the form of an aqueous or anhydrous solution ordispersion, or alternatively the form of an emulsion or suspension.

Thus, the therapeutic agent may be formulated for parenteraladministration (e.g., by injection, for example, bolus injection orcontinuous infusion) and may be presented in unit dose form in ampules,pre-filled syringes, small volume infusion containers or in multi-dosecontainers with an added preservative. The active ingredients may takesuch forms as suspensions, solutions, or emulsions in oily or aqueousvehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredients may be in powder form, obtained by aseptic isolation ofsterile solid or by lyophilization from solution, for constitution witha suitable vehicle, e.g., sterile, pyrogen-free water, before use.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual aerosol dose of each dosage formneed not in itself constitute an effective amount for treating theparticular indication or disease since the necessary effective amountcan be reached by administration of a plurality of dosage units.Moreover, the effective amount may be achieved using less than the dosein the dosage form, either individually, or in a series ofadministrations.

The pharmaceutical formulations of the present invention may include, asoptional ingredients, pharmaceutically acceptable carriers, diluents,solubilizing or emulsifying agents, and salts of the type that arewell-known in the art. Specific non-limiting examples of the carriersand/or diluents that are useful in the pharmaceutical formulations ofthe present invention include water and physiologically acceptablebuffered saline solutions such as phosphate buffered saline solutions pH7.0-8.0. saline solutions and water.

The invention will now be illustrated by the following non-limitingExample.

Example 1 Rational Design of Therapeutic siRNAs: MinimizingOff-Targeting Potential to Improve the Safety of RNAi Therapy forHuntington's Disease

RNA interference (RNAi) provides an approach for the treatment of manyhuman diseases. However, the safety of RNAi-based therapies can behampered by the ability of small inhibitory RNAs (siRNAs) to bind tounintended mRNAs and reduce their expression, an effect known asoff-target gene silencing. Off-targeting primarily occurs when the seedregion (nucleotides 2-8 of the small RNA) pairs with sequences in3′-UTRs of unintended mRNAs and directs translational repression anddestabilization of those transcripts. To date, most therapeutic RNAisequences are selected primarily for gene silencing efficacy, and laterevaluated for safety. Here, in designing siRNAs to treat Huntington'sdisease (HD), a dominant neurodegenerative disorder, we prioritizedselection of sequences with minimal off-targeting potentials (i.e. thosewith a scarcity of seed complements within all known human 3′-UTRs). Weidentified new promising therapeutic candidate sequences which showpotent silencing in cell culture and mouse brain. Furthermore, wepresent microarray data demonstrating that off-targeting issignificantly minimized by using siRNAs that contain “safe” seeds, animportant strategy to consider during pre-clinical development ofRNAi-based therapeutics.

RNAi directs sequence-specific gene silencing by double-stranded RNA(dsRNA) which is processed into functional small inhibitory RNAs(˜21nt). In nature, RNAi for gene regulation occurs primarily via smallRNAs known as microRNAs (miRNAs). Mature microRNAs (˜19-25 nts) areprocessed from larger primary miRNA transcripts (pri-miRNAs) whichcontain stem-loop regions. Via a series of processing events catalyzedby the ribonucleases, Drosha and Dicer, the miRNA duplex region isliberated, and a single strand (the antisense “guide” strand) is thenincorporated into the RNA Induced Silencing Complex (RISC), thusgenerating a functional complex capable of base-pairing with andsilencing transcripts by various means depending on the degree ofcomplementarity. A high-degree of base-pairing causes target transcriptcleavage, whereas imperfect binding (typically to transcript 3′-UTRs)induces the canonical miRNA-based repression mechanism resulting intranslational repression and mRNA destabilization. Indeed for thelatter, pairing via the seed region with as few as 6-7 bp may besufficient to trigger silencing.

Elucidating the mechanisms involved in endogenous miRNA biogenesis andgene silencing has enabled scientists to devise strategies to co-opt thecellular RNAi machinery and direct silencing of virtually any gene ofinterest using siRNAs, short-hairpin RNAs (shRNAs), and artificialmiRNAs; the latter two represent expressed stem-loop transcripts whichrelease siRNAs upon processing. siRNAs are generally designed with theguide strand exhibiting perfect complementarity to the intended mRNAtarget to promote cleavage. This potent gene silencing approach hasbecome a powerful molecular tool to study gene function and is beingdeveloped as a therapeutic strategy to suppress disease-causing genes.The utility of siRNA-based technologies as biological or clinicalinterventions is largely limited by our abilities to design effectiveand specific inhibitory RNAs and to introduce them into target cells ortissues. A major consideration for gene silencing applications isspecificity, and there is mounting evidence supporting that siRNAs bindto and repress unintended mRNAs, an effect known as off-targetsilencing. Off-targeting primarily occurs when the seed region pairswith 3′-UTR sequences in mRNAs and directs translational repression anddestabilization of those transcripts. Recent data supports thatseed-based off-targeting may induce toxic phenotypes. It has beenobserved that the magnitude of siRNA off-targeting is directly relatedto the frequency of seed complements (hexamers) present in the3′-UTRome. By evaluating subsets of siRNAs with differing off-targetingpotentials (low, medium and high; predicted based on hexamerdistributions in human 3′-UTRs), they discovered that siRNAs in the lowsubset had significantly diminished off-target signatures (based onmicroarray data) and less adverse effects on cell viability as comparedto siRNAs in the medium and high subsets. These observations establishedthe importance of considering seed complement hexamer distributions as akey criterion for designing highly specific siRNAs, and some siRNAdesign tools have since incorporated seed-specificity guidelines intotheir algorithms. However, most publically available algorithms remainstrongly biased for gene silencing efficacy over specificity, and thus,very few candidate siRNAs actually contain seeds with low off-targetingpotentials. This is revealed in a literature survey of siRNAs undertherapeutic development; only 7 of 80 recently published siRNAs withtherapeutic relevance (Table 6) could be classified into the lowoff-targeting subgroup. This is problematic as siRNAs move intoearly-stage clinical trials. While potency-based design is rational,current publicly available tools identify only a fraction of thefunctional siRNAs for a given target transcript, and often times, highlyfunctional siRNAs do not satisfy several design rules. For thesereasons, and in the interest of improving the safety profile oftherapeutic RNAi, the inventors hypothesized that a siRNA design schemeprioritizing specificity yet promoting efficacy would yield candidatesiRNA sequences with minimal off-targeting potential and a robustcapacity for potent gene silencing.

Results

Some Artificial miRNAs Induce Sequence-Specific Toxicity

Previous studies from our laboratory and others' have demonstrated thepotential of RNAi therapeutics for treating Huntington's disease (HD), adominant neurodegenerative disease caused by a CAG repeat expansionwhich confers a toxic gain-of-function to the resulting huntingtin (htt)protein. In several rodent models for HD, viral-based expression of RNAihairpins targeting mutant htt mRNA in brain reduced transcript andprotein levels by ˜50-70%, improving behavioral and neuropathologicalphenotypes. Following these proof-of-concept successes, the inventorsinitiated studies to evaluate and optimize the safety of RNAi-basedtherapeutics. The inventors compared the silencing efficacy and safetyof shRNA and artificial miRNA expression vectors in vitro and in vivo.The inventors found that shRNAs are more potent but induce toxicity incell cultures and in mouse brain, whereas artificial miRNAs areexpressed at tolerably lower levels and display better safety profileswhile maintaining potent gene silencing. Since this discovery, theinventors have tested several artificial miRNA sequences in mouse brainusing recombinant adeno-associated viruses (AAV serotype 2/1) fordelivery, and in some instances, have observed sequence-dependenttoxicity. For example, one artificial miRNA targeting htt (miHD-Ex1)caused a high incidence of seizures and morbidity in treated mice (datanot shown); of note, this toxic phenotype was not a consequence of httknockdown, as it has been previously reported that silencing endogenoushtt in mouse brain is tolerated. In another instance, a non-targetedartificial miRNA (miSCR, a scrambled control) induced evidentneurotoxicity as indicated by increased staining for Iba1, a marker forresting and reactive microgila, in treated regions of the striatum (FIG.1). Together, these data suggest that although artificial miRNAs showimproved safety over shRNAs, sequence-dependent toxicity remains aconcern. The inventors therefore explored supplemental means to improvesafety by employing a rational siRNA design scheme intended to minimizethe probability for off-target silencing.

Selection and Screening of Htt-Targeting siRNAs with Low Off-TargetingPotentials

The siRNA toxicity potentials have been correlated with seed complementfrequencies in the human 3′-UTRome (Anderson, E. M., A. Birmingham, S.Baskerville, A. Reynolds, E. Maksimova, D. Leake, et al. (2008).Experimental validation of the importance of seed complement frequencyto siRNA specificity. RNA 14(5):853-61). Here, the inventors estimatedthe number of potential off-target transcripts (POTs) for each hexamerby determining the number of human RefSeq 3′-UTRs containing a specifiedhexamer (out of the 4096 possible). Similar to the previous findings,the majority of hexamers are present in ˜4000 3′-UTRs or more, andinterestingly, there is an unexplainable peak (containing 1135 hexamers)in the distribution. These latter hexamers are present in less than 20003′-UTRs (FIG. 2c ). Since siRNAs are typically designed to target codingregions, we determined the probability of finding these relatively rarehexamers in human RefSeq coding exons. This was ˜14%, suggesting that 1in 7-8 randomly designed siRNAs would contain these rare hexamers in theseed region. To improve upon this nominal probability, low frequencyhexamers may first be located within target transcript sequence andsubsequently used as a foundation for designing siRNAs with minimaloff-targeting potentials. For example, the inventors scanned the humanhtt coding sequence for low frequency hexamers, and with each instance,examined the nearby context to determine whether the siRNA containingthe hexamer seed complement would satisfy two criteria: (1) faithfulloading of the intended antisense guide strand and (2) GC-contentbetween 20-70% (FIG. 3a ). Not only do these attributes represent themost prominent determinants of siRNA potency, but proper loading of theantisense guide strand is mandated to mitigate potential off-targetingmediated by the sense “passenger” strand. Strand-loading is dictated bythe thermodynamic properties present at the siRNA duplex ends, withguide strand loading encouraged by weak pairing (A/G-U) at the 5′ endand strong G-C binding at the opposing terminus (FIG. 3a ). Of note, theinventors apply this principle to the terminal two base-pairs at eachend and take advantage of weak G-U wobble pairing to impart instabilityat the 5′ end of the antisense strand when applicable. Finally, theinventors select siRNAs based on a fairly liberal range of GC-content(20-70%) which supports a suitable potential for efficient silencing(>80%), as determined by our evaluation of large-scale knock-down datafor 2431 randomly designed siRNAs targeting 31 unique mRNAs (data notshown). As with most siRNA design algorithms, candidate siRNA sequencessatisfying the above criteria are subjected to BLAST to evaluate thepotential for off-target cleavage events mediated by near-perfectcomplementarity to unintended mRNAs (for BLAST parameters, seeBirmingham, A., E. Anderson, K. Sullivan, A. Reynolds, Q. Boese, D.Leake, et al. (2007). A protocol for designing siRNAs with highfunctionality and specificity. Nat Protoc 2(9):2068-78)).

Using the inventors' siRNA design criteria (low POTs seed,strand-biasing, and GC-content), the inventors initially identifiedeight htt-targeting candidate sequences for further testing. We embeddedthe siRNA sequences into the context of the inventors' U6-drivenartificial miRNA-based expression vectors (FIG. 2a ) and screened themfor gene silencing against endogenous htt in HEK293 cells (FIG. 3b ).The inventors observed two candidates (HDS1 and HDS2, Tables 3 and 4,and FIGS. 7 and 8) that effectively silence htt mRNA (˜50%, relative tocontrol). Notably, this magnitude of in vitro silencing againstendogenous htt is comparable to the levels achieved by other htt RNAisequences (including HD2.4) that previously showed therapeutic efficacyin HD mouse models (Harper, S. Q., P. D. Staber, X. He, S. L. Eliason,I. Martins, Q. Mao, et al. (2005). RNA interference improves motor andneuropathological abnormalities in a Huntington's disease mouse model.Proceedings of the National Academy of Sciences, USA 102(16):5820-5825;Rodriguez-Lebron, E., E. M. Denovan-Wright, K. Nash, A. S. Lewin, and R.J. Mandel (2005). Intrastriatal rAAV-mediated delivery ofanti-huntingtin shRNAs induces partial reversal of disease progressionin R6/1 Huntington's disease transgenic mice. Mol Ther 12(4):618-633;Boudreau, R. L., J. L. McBride, I. Martins, S. Shen, Y. Xing, B. J.Carter, et al. (2009). Nonallele-specific silencing of mutant andwild-type huntingtin demonstrates therapeutic efficacy in Huntington'sdisease mice. Mol Ther 17(6):1053-63).

Microarray Analyses of Seed-Related Off-Targeting

To validate the low off-targeting potential of these effective sequences(HDS1 and HDS2) and the inventors' siRNA design scheme, the inventorsperformed microarray analysis to assess seed-related off-target genesilencing. The inventors included several RNAi constructs which targethuman htt and various control sequences to help discern off-target genesilencing from gene expression changes that result from suppressing htt(Table 1). Of note, all sequences used were designed to promote properloading of the antisense strand to avoid the confounding potential ofoff-targeting mediated by the passenger strand. The htt-silencing groupconsisted of HDS1, HDS2, HD2.4 and HD8.2; the latter two were previouslydesigned without regard for the seed sequence and have >4500 POTs each(FIG. 2c ). The control group (i.e. non-htt-targeting) consisted ofseveral sequences (8.2 mis, 8.2-124a, Terror, and Safe), each designedto serve a unique purpose (Table 1). 8.2 mis contains the same seed asHD8.2 but has central mismatches to prevent htt silencing, while8.2-124a and Terror are HD8.2 scrambled sequences which respectivelycontain a seed mimic of miR-124a (a naturally occurring and highlyconserved miRNA) and a seed with high off-targeting potential (i.e.complements a highly abundant hexamer in the human 3′-UTRome). Of note,8.2-124a was included as a control for detecting seed-relatedoff-targeting within the microarray data and to underscore theprospective concern of designing siRNAs (scrambled controls or on-targetsequences) such that they unintentionally contain naturally occurringmiRNA seeds. Finally, the Safe construct contains an arbitrary sequencedesigned to have low off-targeting potential based on 3′-UTR hexamerfrequencies. Together, these constructs provide a wide-range ofoff-targeting potentials and address problems that can inadvertentlyarise when including scrambled sequences as controls in RNAiexperiments, a commonly used practice.

The inventors carried out transcriptional profiling in cultured HEK293cells 72 h after transfection with RNAi expression plasmids (N=4 perconstruct). Initially, gene expression changes were detected byperforming ANOVA statistical analysis using all treatments included inthe study. As anticipated, htt was consistently among the mostsignificantly down-regulated transcripts in samples treated withhtt-targeting RNAi sequences (P<5e-11, relative to U6), and thesemicroarray data were corroborated by QPCR evaluation of htt mRNA levelsin the same RNA samples (FIG. 4a ). Next, the inventors performedhierarchical clustering using differentially expressed genes within thedataset (P<0.0001, 827 genes) to measure the relatedness among thevarious treatments. These include gene expression changes which occur asa result of knocking down endogenous htt in addition tosequence-specific off-targeting events. Notably, we observed a closerrelationship between the low off-targeting potential sequences (Safe,HDS1 and HDS2) and the U6 promoter-only control as compared to theremaining sequences, which were designed either blindly (HD2.4 andHD8.2) or intentionally with mid-to-high off-targeting potentials(8.2-124a and Terror). These clustering results support a clearassociation between off-targeting potential and impact on thetranscriptional profile (FIG. 4b ), corroborating the Anderson et al.observations (Anderson, E. M., A. Birmingham, S. Baskerville, A.Reynolds, E. Maksimova, D. Leake, et al. (2008). Experimental validationof the importance of seed complement frequency to siRNA specificity. RNA14(5):853-61). In addition, these data substantiate that changes relatedto off-targeting are more robust than those resulting from httsilencing. Visualization of the complementing heatmap made obvious theoverwhelming amount of off-targeting caused by Terror and, to a slightlylesser degree, 8.2-124a (FIG. 4b ). The overlap between these sequencesis likely due to their seed similarity (Table 1), and subsequentanalyses confirmed that much of this off-targeting was seed-related. Forthe sequences with low-to-mid off-targeting potentials, the relationshipbetween off-targeting potentials and gene expression profiles was bettervisualized by removing the Terror and 8.2-124a samples from the ANOVAanalysis and repeating hierarchical clustering of differentiallyexpressed genes (P<0.01, 985 genes) (FIG. 4c ). With this approach, theheat maps showed gene suppression signatures that were unique to each ofthese sequences, with the exception of HD8.2 and 8.2 mis. As previouslynoted, these constructs share the same seed sequence, and this evidentoverlap affirms that much of the observed gene expression changes areseed-related, rather than caused by htt knockdown. In addition, thisexample highlights the benefit of designing on-target and control siRNAsequences that share the same seed. This preserves off-targeting betweenthe two sequences and is therefore beneficial when applying RNAi-basedtools to study gene function or validate drug targets.

The inventors next assessed whether the observed gene expression changescould be explained by seed-mediated gene silencing. Cumulativedistribution analyses of gene expression levels indicated thattranscripts containing seed binding sites for the antisense strand intheir 3′-UTR had a much higher probability of being down-regulated (i.e.curve shifting left) (FIG. 4d , top and FIG. 6), and the degree ofdown-regulation was dependent upon the number of binding sites present,consistent with previous reports characterizing miRNA seed-mediatedsilencing of target transcripts. The inventors also performed cumulativefraction analyses to detect seed-related gene silencing caused by thepassenger strand; in this case, the presence of 3′-UTR binding sites hadlittle to no detectable influence on gene expression, supporting thatthe current vector design (i.e. two strong G-C base-pairs at the sense5′ and two weak A/G-U base-pairs at the sense 3′) promotes properstrand-biasing (FIG. 4d , bottom). As a complementary approach to detectseed-related gene silencing events, the inventors performed motifdiscovery analyses using 3′-UTR sequences of down-regulated transcriptsunique to each treatment group. In all instances, the inventors foundsignificant enrichment of motifs complementary to the respective seedsequences in the uniquely down-regulated transcript 3′-UTRs relative toa background 3′-UTR dataset consisting of all known human 3′-UTRs (FIG.4e and FIG. 6b ). These data confirm that seed-related off-target genesilencing is present in the datasets for all RNAi sequences tested. Uponfurther evaluation, the inventors estimated the number of seed-relatedoff-targets for each RNAi sequence by identifying transcripts that weredown-regulated (1.1-fold, P<0.05, relative to U6) and contain therelevant seed complements in their 3′-UTR (Table 2). This analysisrevealed that using the present “safe” seed design method, HDS1 and HDS2show nearly a log improvement in minimizing seed-related off-targeting,as compared to previous lead candidates, HD2.4 and HD8.2.

In Vivo Silencing and Safety of HDS Sequences

Having identified that HDS1 and HDS2 have substantially fewerseed-related off-targets, the inventors next tested these sequences forsilencing and safety in vivo in mouse brain. The inventorsintrastriatally injected AAV1-miHDS1, AAV1-miHDS2 or AAV1-miSafe(control) into two cohorts of wild-type mice. Of note, HDS1 exhibitsfull complementarity to mouse, rhesus and human htt sequences, making itan attractive candidate for preclinical testing. HDS2 only targets humanhtt, with mismatches to the corresponding mouse and rhesus targetsequences. At three weeks post-injection, the inventors performed QPCRanalyses to evaluate gene silencing efficacy in striatal tissueharvested from the first cohort of animals and observed significant httmRNA knockdown (˜60%) in mice treated with AAV1-miHDS1, relative touninjected and AAV1-miSafe-treated mice (FIG. 5a ). Notably, previousreports from the inventors' laboratory and others' demonstrate that ˜60%silencing of striatal htt transcripts in HD mouse models markedlyreduces protein levels, resulting in appreciable therapeutic efficacy.The second cohort of mice was sacrificed at six months post-injection toevaluate long-term vector tolerability. Staining for Iba1, a marker forresting and reactive microgila, showed no evidence for neurotoxicity intransduced regions of the striata, relative to nearby untransducedtissue (FIG. 5b ; refer to FIG. 1 for comparison to miSCR, a toxicsequence with high off-targeting potential). These results areencouraging considering that HD2.4, previously shown to betherapeutically efficacious in short term studies, caused modest butstill detectable increases in Iba1 staining in both wild-type and HDmice. Furthermore, the data corroborate previous reports demonstratingthat reducing wild-type htt mRNA levels by ˜60% in mouse striatum doesnot induce overt neurotoxicity.

DISCUSSION

Although the absolute specificity and safety of RNAi approaches remainsquestionable, recent advances in understanding RNAi-induced toxicities(e.g. pathway saturation and off-targeting) are facilitating researchersin devising strategies to limit these adverse events. For example, thediscovery that high-level shRNA expression causes lethality in mice(Grimm, D., K. L. Streetz, C. L. Jopling, T. A. Storm, K. Pandey, C. R.Davis, et al. (2006). Fatality in mice due to oversaturation of cellularmicroRNA/short hairpin RNA pathways. Nature 441(7092):537-41) promptedus to test alternative hairpin-based vectors (e.g. artificial miRNAs)for their capacity to limit the production of RNAi substrates followingviral-based delivery in vivo, thus resulting in improved tolerability.Furthermore, Anderson et al recently evaluated the impact of 3′-UTR seedcomplement frequencies on siRNA off-targeting potentials, using a set ofrandomly designed siRNA sequences targeting a variety of genes(Anderson, E. M., A. Birmingham, S. Baskerville, A. Reynolds, E.Maksimova, D. Leake, et al. (2008). Experimental validation of theimportance of seed complement frequency to siRNA specificity. RNA14(5):853-61). Low off-targeting potential siRNAs were found to exhibithigher specificity as per mRNA profiling, lower toxicity and fewer falsepositives in phenotypic screens. The authors proposed that siRNAs withlow seed complement frequencies improve the accuracy of RNAi screens tostudy gene function or validate drug targets. Here, the inventors tookadvantage of these findings to deliberately design therapeutic siRNAswith low off-targeting potentials, as a means to promote safety inpre-clinical development of RNAi therapy for HD. The inventorsidentified two candidates (HDS1 and HDS2) which effectively silencehuman htt mRNA, induce minimal seed-related off-targeting and arewell-tolerated in mouse brain long-term.

Although the inventors' work was initially undertaken to develop siRNAswith low off-targeting potentials, a similar strategy may be employed tointentionally design siRNAs with high off-targeting capacities (e.g.Terror sequence) for use as anti-tumor agents. This approach may detertumor escape by more broadly disrupting essential cellular pathways, ascompared to delivering siRNAs targeting specific oncogenes.

Researchers using RNAi triggers in basic and translational researchoften employ scrambled sequences as controls. The present workhighlights the importance of carefully designing control siRNAs, withattention to putative seed sequences that can inadvertently induceconsiderable off-target silencing via miRNA-based mechanisms. Here, theinventors intentionally introduced either a known miRNA seed (8.2-124a)or a seed with high off-targeting potential (Terror) into scrambledsiRNA sequences. As expected, both induced significant seed-relatedalterations in transcriptional profiles, when compared to our controlvector (Safe) designed to exhibit low off-targeting potential.Furthermore, we describe and test the design of a “same seed” controlvector (8.2 mis). This approach resulted in significant preservation ofoff-targeting relative to the corresponding on-target sequence (HD8.2).These data encourage the use of “same seed” controls in future RNAiexperiments.

There are several key considerations which apply to “safe” seed siRNAdesign. First, low off-targeting potential does not necessarily meannon-toxic, as off-target identity remains a crucial influence ontolerability. The inventors' improved ability to accurately identifyhigh probability off-targets allows us to better select lead candidatesiRNAs, particularly when several low off-targeting sequences areavailable for a given target sequence. Second, observed safety inpre-clinical toxicity studies in either rodents or non-human primatesmay not ensure success in humans, as differences in 3′-UTR sequencescreates off-targeting profiles unique to each species (Burchard, J., A.L. Jackson, V. Malkov, R. H. Needham, Y. Tan, S. R. Bartz, et al.(2009). MicroRNA-like off-target transcript regulation by siRNAs isspecies specific. Rna 15(2):308-15). It is important to note, thatalthough off-target identities may be species-specific, theoff-targeting potentials for each hexamer remain highly consistent, ashexamer frequencies among several species (e.g. mouse, rhesus and human)show minimal variability (data not shown). Third, locating these rarehexamers may be difficult in small target transcripts, and thus othermeans to limit off-targeting may be necessary. For instance, severalreports have demonstrated that certain chemical modifications to theseed nucleotides significantly reduce off-targeting from chemicallysynthesized siRNAs (Jackson, A. L., J. Burchard, D. Leake, A. Reynolds,J. Schelter, J. Guo, et al. (2006). Position-specific chemicalmodification of siRNAs reduces “off-target” transcript silencing. RNA12(7):1197-205; Bramsen, J. B., M. M. Pakula, T. B. Hansen, C. Bus, N.Langkjaer, D. Odadzic, et al. (2010). A screen of chemical modificationsidentifies position-specific modification by UNA to most potently reducesiRNA off-target effects. Nucleic Acids Res 38(17):5761-73; Vaish, N.,F. Chen, S. Seth, K. Fosnaugh, Y. Liu, R. Adami, et al. (2011). Improvedspecificity of gene silencing by siRNAs containing unlocked nucleobaseanalogs. Nucleic Acids Res 39(5):1823-32). The prospect of combining“safe” seed design with chemical modifications serves as a provocativestrategy to develop synthetic siRNAs with very high specificity.However, for expressed RNAi, chemical modifications are not applicable,thus “safe” seed design provides the primary means to limitoff-targeting for these hairpin-based vectors.

In summary, “safe” seed siRNA design has significant implications fortherapeutic development which may result in substantial time- andcost-saving opportunities. Traditional small molecules are initiallyscreened for efficacy and later tested for safety, since predictingpotential side effects remains a challenge due to the complex nature ofsmall molecule interactions. By contrast, the inventors' ability topredict off-targeting (derived from base-pairing) foroligonucleotide-based drugs provides a unique opportunity to prioritizesafety during drug development and subsequently screen for efficacy.

Materials & Methods

Plasmids and Viral Vectors

The plasmids expressing mouse U6-driven artificial miRNAs were cloned aspreviously described using the DNA oligonucleotides listed in Table 5(Boudreau, R. L., A. Mas Monteys, and B. L. Davidson (2008). Minimizingvariables among hairpin-based RNAi vectors reveals the potency ofshRNAs. RNA 14:1834-1844). For AAV production, artificial miRNAexpression cassettes were cloned into pFBGR-derived plasmids whichco-express CMV-driven GFP (Boudreau, R. L., I. Martins, and B. L.Davidson (2009). Artificial MicroRNAs as siRNA Shuttles: Improved Safetyas Compared to shRNAs In vitro and In vivo. Mol Ther 17(1):169-17).

Recombinant AAV serotype 2/1 vectors (AAV1-GFP, AAV1-miSCR, AAV1-miHDS1,and AAV1-miHDS2 were generated by the University of Iowa Vector Corefacility as previously described (Urabe, M., C. Ding, and R. M. Kotin(2002). Insect cells as a factory to produce adeno-associated virus type2 vectors. Hum Gene Ther 13(16):1935-1943). Viruses were initiallypurified using an iodixanol gradient (15-60% w/v) and subjected toadditional purification via ion exchange using MustangQ Acrodiscmembranes (Pall Corporation, East Hills, N.Y.). AAV1 vectors wereresuspended in Formulation Buffer 18 (HyClone, Logan, Utah), and titers(viral genomes per ml) were determined by QPCR.

AAV Injections and Brain Tissue Isolation

All animal protocols were approved by the University of Iowa Animal Careand Use Committee. Wildtype FVB mice were injected with AAV1 vectors aspreviously reported (Harper, S. Q., P. D. Staber, X. He, S. L. Eliason,I. Martins, Q. Mao, et al. (2005). RNA interference improves motor andneuropathological abnormalities in a Huntington's disease mouse model.Proceedings of the National Academy of Sciences, USA 102(16):5820-5825;McBride, J. L., R. L. Boudreau, S. Q. Harper, P. D. Staber, A. M.Monteys, I. Martins, et al. (2008). Artificial miRNAs mitigateshRNA-mediated toxicity in the brain: Implications for the therapeuticdevelopment of RNAi. Proc Natl Acad Sci USA 105(15):5868-73). For allstudies, unless indicated otherwise, mice were injected bilaterally intothe striatum (coordinates: 0.86 mm rostral to bregma, ±1.8 mm lateral tomidline, 3.5 mm ventral to the skull surface) with 4 ul of AAV1 virus(at ˜1×10¹² viral genomes/ml). Mice used in histological analyses wereanesthetized with a ketamine/xylazine mix and transcardially perfusedwith 20 ml of 0.9% cold saline, followed by 20 ml of 4% paraformaldehydein 0.1M PO₄ buffer. Mice were decapitated, and the brains were removedand post-fixed overnight in 4% paraformaldehyde. Brains were stored in a30% sucrose solution at 4° C. until cut on a sliding knife microtome at40 μm thickness and stored at −20° C. in a cryoprotectant solution. Miceused for QPCR analyses were perfused with 20 ml of 0.9% cold saline.Brains were removed and sectioned into 1 mm thick coronal slices using abrain matrix (Roboz, Gaithersburg, Md.). Tissue punches were taken fromthe striatum using a tissue core (1.4 mm in diameter) and triterated in50 ul of TRIzol (Invitrogen, Carlsbad, Calif.). RNA was isolated fromstriatal punches using 1 ml of TRIzol.

Immunohistochemical Analyses

Free-floating, coronal brain sections (40 μm thick) were processed forimmunohistochemical visualization of microglia (anti-Iba1, 1:1000, WAKO,Richmond, Va.). All staining procedures were carried out as previouslydescribed (McBride, J. L., R. L. Boudreau, S. Q. Harper, P. D. Staber,A. M. Monteys, I. Martins, et al. (2008). Artificial miRNAs mitigateshRNA-mediated toxicity in the brain: Implications for the therapeuticdevelopment of RNAi. Proc Natl Acad Sci USA 105(15):5868-73), using goatanti-rabbit IgG secondary antibody (1:200) and Vectastain ABC-peroxidasereagent (both from Vector Laboratories, Burlingame, Calif.). Stained orunstained (the latter for visualization of GFP autofluorescence)sections were mounted onto Superfrost Plus slides (Fisher Scientific,Pittsburgh, Pa.) and coverslipped with Gelmount (Biomeda, Foster City,Calif.) or Vectashield (Vector Laboratories). Images were captured usingan Olympus BX60 light microscope and DP70 digital camera, along withOlympus DP Controller software (Olympus, Melville, N.Y.).

Hexamer Distribution Analyses

All human RefSeq IDs, official gene symbols, and coding and 3′-UTRsequences (Hg19, GRCH37) were obtained and only sequences with NM_*pre-fixes were used for analysis. For 3′-UTR sequences, thenon-overlapping frequency of each individual hexamer (4096 possible) wascounted to determine the number of 3′-UTRs containing a given hexamer.Non-overlapping sites were considered to account for actual binding siteavailability. For coding sequence, the total hexamer frequencies weredetermined, allowing overlapping hexamers, to estimate the probabilityof selecting siRNA sequences containing the specified hexamer. For geneswith variants (i.e. same official gene symbol but different accessionnumber), the maximum count for each hexamer was used.

Cell Culture and Transfection

For the HDS screen, HEK293 cells were grown in 24-well plates in growthmedia containing 10% fetal bovine serum (FBS) and transfected inquadruplicate with 400 ng of plasmid using Lipofectamine 2000(Invitrogen) by adding the lipid:DNA complexes directly to the growthmedia. Total RNA was isolated at 24 h post-transfection using 1 ml ofTrizol. For microarray studies, HEK293 cells were grown in 12-wellplates in growth media (10% FBS) and transfected with 1 ug of plasmidunder serum-free conditions. Lipid:DNA complexes were removed 3 h laterand replaced with growth media (5% FBS). At 72 h (microarray)post-transfection, total RNA was isolated using 1 ml of TRIzol.

Quantitative Real-Time PCR (QPCR)

Random-primed first-strand cDNA synthesis was performed using 500 ngtotal RNA (High Capacity cDNA Reverse Transcription Kit; AppliedBiosystems, Foster City, Calif.) per manufacturer's protocol. Assayswere performed on a sequence detection system using primers/probe setsspecific for human htt and GAPDH or mouse htt and beta-actin (Prism7900HT and TaqMan 2× Universal Master Mix; Applied Biosystems). Relativegene expression was determined using the ΔΔC_(T) method, normalizing toeither GAPDH or beta-actin mRNA levels.

Microarray Analyses

Microarray analysis was done with assistance from the University of IowaDNA Facility (Iowa City, Iowa). Fifty nanograms of total RNA templatewere used to produce amplified cDNA using the Ovation Biotin RNAAmplification System, v2 (NuGEN Technologies) following themanufacturer's protocol. Amplified cDNA product was purified with DNAClean and Concentrator-25 (Zymo Research). 3.75 μg of amplified cDNAwere processed using the FL-Ovation cDNA Biotin Module v2 (NuGENTechnologies, San Carlos, Calif.) to produce biotin labeled antisensecDNA in 50- to 100 bp fragments. Following denaturation at 99° C. for 2min, fragmented, labeled cDNA were combined with hybridization controloligomer (b2) and control cRNAs (BioB, BioC, BioD, and CreX) inhybridization buffer and hybridized to the HuGene 1.0ST GeneChip(Affymetrix, Santa Clara, Calif.) capable of detecting more than 28,000genes. Following an 18 hour incubation at 45° C., the arrays werewashed, stained with streptavidinphycoerythrin (Molecular Probes), andthen amplified with an anti-streptavidin antibody (Vector Laboratories)using the Fluidics Station 450 (Affymetrix). Arrays were scanned withthe Affymetrix Model 3000 scanner and data collected using GeneChipoperating software (GCOS) v1.4. Each sample and hybridization underwenta quality control evaluation, including percentage of probe setsreliably detecting between 40 and 60% present call and 3′-5′ ratio ofthe GAPDH gene less than 3.

Partek Genomics Suite (Partek GS, Saint Louis, Mo.) was used topreprocess, normalize and analyze microarray data. Affymetrix array rawfluorescence intensity measures of gene expression were normalized andquantified using robust multi-array analysis (RMA). To identifydifferentially expressed genes among the nine treatment groups (N=4each, Table 1), the inventors employed two-way ANOVA (variables: scandate and treatment) since arrays were processed in groups of four (onereplicate per treatment in each group). Pair-wise contrasts betweengroups of interest were performed when indicated. Principal componentand hierarchical clustering analyses were used to visualize differentialgene expression.

Cumulative Distribution Analyses

3′-UTR sequences for all RefSeq mRNAs on the HuGene 1.0 ST chip wereobtained, and the number of non-overlapping seed complement bindingsites (octamers) per 3′-UTR for each of the indicated inhibitory RNAswas determined. Three possible octamers for each artificial miRNA wereconsidered to account for flexibility in Drosha and Dicer cleavage(Table 5). Transcripts were parsed into groups depending on the numberof seed complements in their 3′-UTR (no sites, 1 site, 2+ sites), andcumulative distributions of gene expression values (Log 2 fold-change,relative to U6) were plotted. Two-sample Kolmogorov-Smirnov (KS) testswere performed to evaluate the statistical significance ofdistributional deviations relative to baseline (no sites).

Motif Discovery

The Venn diagram feature on Partek GS was used to create lists ofuniquely down-regulated genes (1.1-fold, P<0.05 or 1.2-fold, P<0.01,relative to U6) for each treatment, taking into account htt silencing(e.g. HDS1, HDS2, HD2.4 and HD8.2 were included in one Venn diagram, andSafe, Terror, 8.2 mis and 8.2-124a were included in another). EnsemblGene IDs were obtained using the Gene ID Conversion Tool at the DavidBioinformatics Resources web-server (Huang da, W., B. T. Sherman, Q.Tan, J. R. Collins, W. G. Alvord, J. Roayaei, et al. (2007). The DAVIDGene Functional Classification Tool: a novel biological module-centricalgorithm to functionally analyze large gene lists. Genome Biol8(9):R183). Ensemble Gene IDs were subjected to target set analysisusing the Amadeus Motif Discovery Platform (Allegro Software Package) toidentify 8mers enriched in the target set 3′-UTRs, relative the providedhuman 3′-UTR background dataset (Halperin, Y., C. Linhart, I. Ulitsky,and R. Shamir (2009). Allegro: analyzing expression and sequence inconcert to discover regulatory programs. Nucleic Acids Res37(5):1566-79). Amadeus blindly identified an enrichment of seedcomplement motifs for each RNAi sequence tested, and the lowest p-valuesfor the relevant motifs were reported.

TABLE 1 Microarray constructs. Off-Targeting Targets 8mer PotentialPurpose/Design Construct HTT? Seed (# of OTs*) Rationale U6 promoter NoN/A N/A Normalizing control mHDS1 Yes GUCGACCA Low New lead candidate (495) containing safe seed miHDS2 Yes AUAGUCGC Low New lead candidate(1227) containing safe seed miHD2.4 Yes UAGACAAU Mid Previous candidate(4688) selected at random miHD8.2 Yes AUAAACCU Mid Previous candidate(5041) selected at random mi8.2mis No AUAAACCU Mid “Same seed” control(5041) for 8.2 sequence mi8.2-124a No UAAGGCAC Mid-HighScrambled 8.2 sequence (5519) containing miR-124a seed miTerror NoAAGGCAGA High Scrambled 8.2 sequence (7218) containing toxic seedsmiSafe No AAACGCGU Low Random sequence with   (662)minimal off-targeting *Average number of transcripts containing seedhexamer complements. Three possible hexamers were considered for each8mer seed to account for flexibility in Drosha/Dicer processing.

TABLE 2 Off-target summary. Sequence # of Off-targets* Avg. Fold Δ HD8.279 −1.17 HD2.4 73 −1.17 HDS1 7 −1.27 HDS2 12 −1.17 Safe 9 −1.18 Terror450 −1.26 (*Down-regulated genes with 8mer seed complement in 3′-UTR)

TABLE 3 miHDS sequences that effectively silence endogenous htt mRNA inHEK293 cells (human-derived) Predicted Predicted Silencing antisenseSpecificity Artificial Pri-miRNA RNA Human Rhesus Mouse miRNA Sequencesequence #1 (exon) (exon) (exon) miHDS.1 5′...cucgagu 5′- Yes Yes Yesgagcgaugcugg  gucgaccaugcg (44) (51) (44) cucgcauggucg agccagcac-3′auacuguaaagc SEQ ID NO: 4 cacagaugggug ucgaccaugcga gccagcaccgccuacuaga...3′ SEQ ID NO: 1 miHDS.2 5′...cucgagu 5′- Yes No Nogagcgcucccgg auagucgcugau (61) ucaucagcgacu gaccgggau-3′ auuccguaaagcSEQ ID NO: 5 cacagaugggga uagucgcugaug accgggaucgcc uacuaga...3′SEQ ID NO: 2 miHDS.5 5′...cucgagu 5′- Yes No No gagcgcuccucuuuacgucguaaa (3′UTR- uguuuacgacgu caagaggaa-3′ long) gaucuguaaagcSEQ ID NO: 6 cacagaugggau uacgucguaaac aagaggaacgcc uacuagu...3′SEQ ID NO: 3

TABLE 4 miHDS sequences that effectively silence endogenous htt mRNA inHEK293 cells (human-derived) Artificial miRNAFull-length Pri-miRNA Sequence miHDS.15′-GCGUUUAGUGAACCGUCAGAUGGUACCGUUU AAACUCGAGUGAGCGAUGCUGGCUCGCAUGGUCGAUACUGUAAAGCCACAGAUGGGUGUCGACCAUGC GAGCCAGCACCGCCUACUAGAGCGGCCGCCACAGCGGGGAGAUCCAGACAUGAUAAGAUACAUU-3′ SEQ ID NO: 10 miHDS.25′-GCGUUUAGUGAACCGUCAGAUGGUACCGUUU AAACUCGAGUGAGCGCUCCCGGUCAUCAGCGACUAUUCCGUAAAGCCACAGAUGGGGAUAGUCGCUGA UGACCGGGAUCGCCUACUAGAGOGGCCGCCACAGCGGGGAGAUCCAGACAUGAUAAGAUACAUU-3′ SEQ ID NO: 11 Pri-5′-CUCGAGUGAGCGAUGCUGGCUCGCAUGGUCG miHDS.1AUACUGUAAAGCCACAGAUGGGUGUCGACCAUGC GAGCCAGCACCGCCUACUAGA-3′SEQ ID NO: 33

TABLE 5 Artificial miRNA Sequences (SEQ ID Nos: 75-119, respectively, inorder of appearance) miHDS1

Oligo 1: aaaactcgagtgagcgatgctggctcgcatggtcgatactgtaaagccacagatgggOligo 2: aaaaactagtaggcggtgctggctcgcatggtcgacacccatctgtggctttacagCumulative Distribution Antisense Seed Complements: ATGGTCGA, TGGTCGAC, GGTCGACA miHDS2

Oligo 1: aaaactcgagtgagcgctcccggtcatcagcgactattccgtaaagccacagatggOligo 2: aaaaactagtaggcgatcccggtcatcagcgactatccccatctgtggctttacagCumulative Distribution Antisense Seed Complements: AGCGACTA, GCGACTAT, CGACTATC miHDS3

Oligo 1: aaaactcgagtgagcggtgcttctttgtcagcgcgtttccgtaaagccacagatgggOligo 2: aaaaactagtaggcgctgcttctttgtcagcgcgtcccccatctgtggctttacag miHDS4

Oligo 1: aaaactcgagtgagcgacggggcagcaggagcggtagactgtaaagccacagatgggOligo 2: aaaaactagtaggcggcggggcagcaggagcggtaaacccatctgtggctttacag miHDS5

Oligo 1: aaaactcgagtgagcgctcctcttgtttacgacgtgatctgtaaagccacagatgggOligo 2: aaaaactagtaggcgttcctcttgtttacgacgtaatcccatctgtggctttacag miHDS6

Oligo 1: aaaactcgagtgagcgcgggatgtagagaggcgttagtctgtaaagccacagatgggOligo 2: aaaaactagtaggcgtgggatgtagagaggcgttaatcccatctgtggctttacag miHDS7

Oligo 1: aaaactcgagtgagcgccccttggaatgcatatcgttgctgtaaagccacagatgggOligo 2: aaaaactagtaggcgtcccttggaatgcatatcgctacccatctgtggctttacag miHDS8

Oligo 1: aaaactcgagtgagcgcacgtggacctgcctacggaggccgtaaagccacagatgggOligo 2: aaaaactagtaggcgaacgtggacctgcctacggaaacccatctgtggctttacagmiHD2.4

Oligo 1: aaaactcgagtgagcgcaccgtgtgaatcattgtctaactgtgaagccacagatgggOligo 2: aaaaactagtaggcgtaccgtgtgaatcattgtctaacccatctgtggctttacagCumulative Distribution Anitsense Seed Complements: CATTGTCT, ATTGTCTA, TTGTCTAA miHD8.2

Oligo 1: aaaactcgagtgagcgaagcagcttgtccaggtttatgctgtgaagccacagatgggOligo 2: aaaaactagtaggcggagcagcttgtccaggtttatacccatctgtggctttacagCumulative Distribution Antisense Seed Complements: CAGGTTTA, AGGTTTAT, GGTTTATA mi8.2mis

Oligo 1: aaaactcgagtgagcgaagcagctgtgttaggtttatgctgtgaagccacagatgggOligo 2: aaaaactagtaggcggagcagctgtgttaggtttatacccatctgtggctttacagCumulative Distribution Antisense Seed Complements: TAGGTTTA, AGGTTTAT, GGTTTATA mi8.2-124a

Oligo 1: aaaactcgagtgagcgaagctgtagctatgtgccttagctgtgaagccacagatgggOligo 2: aaaaactagtaggcggagctgtagctatgtgccttaacccatctgtggctttacagCumulative Distribution Antisense Seed Complements: TGTGCCTT, GTGCCTTA, TGCCTTAA miTerror

Oligo 1: aaaactcgagtgagcgcagcaggagttattctgccttactgtaaagccacagatgggOligo 2: aaaaactagtaggcgtagcaggagttattctgccttacccatctgtggctttacagCumulative Distribution Antisense Seed Complements: TTCTGCCT, TCTGCCTT, CTGCCTTA miSafe

Oligo 1: aaaactcgagtgagcgcagcgaacgacttacgcgtttactgtaaagccacagatgggOligo 2: aaaaactagtaggcgtagcgaacgacttacgcgtttacccatctgtggctttacagCumulative Distribution Antisense Seed Complements: TACGCGTT, ACGCGTTT, CGCGTTTA miSCR

Oligo 1: aaaactcgagtgagcgcaccatcgaaccgtcagagttactgtgaagccacagatgggOligo 2: aaaaactagtaggcgtaccatcgaaccgtcagagttacccatctgtggctttacag

TABLE 6 siRNA Literature Survey Antisense Sequence 2-7 3-8(SEQ ID NOs: 120-199, SC SC respectively, in order 2-7 Seed 3-8 Seed #of # of of appearance) Complement Complement OTs OTs Target ReferenceTTCGATCTGTAGCAGCAGCTT GATCGA AGATCG  629 1104 HTT  [1]GATCCGACTCACCAATACC TCGGAT GTCGGA  651  617 bcl-xl  [2]TTCCGAATAAACTCCAGGCTT TTCGGA ATTCGG  937  704 PCSK9  [3]ACGTAAACAAAGGACGTCC TTTACG GTTTAC  995 4054 HBV  [4]AACGTTAGCTTCACCAACATT TAACGT CTAACG 1112  668 c-myc  [5]TAACGTAACAGTCGTAAGA TACGTT TTACGT 1193 1220 bim  [6] ACAGCGAGTTAGATAAAGCTCGCTG CTCGCT 1505 1671 c-myc  [7] CACACGGGCACAGACTTCCAA CCGTGT CCCGTG2017 2023 HTT  [1] AGGTGTATCTCCTAGACACTT TACACC ATACAC 2330 3366 PCSK9 [3] TGTGCTACGTTCTACGAG TAGCAC GTAGCA 2828 3383 HCV  [8]TGTGGACAAAGTCTCTTCC GTCCAC TGTCCA 2930 4899 Livin  [9]TGATGTCATAGATTGGACT GACATC TGACAT 3143 5012 CCR5 [10]TCTGATCTGTAGCAGCAGCTT GATCAG AGATCA 3261 4214 HTT  [1]GGTAAGTGGCCATCCAAGC ACTTAC CACTTA 3268 4049 bcl-xl  [2]CGAGTTAGATAAAGCCCCG TAACTC CTAACT 3319 3265 c-myc  [7]TTAACCTAATCTCCTCCCC AGGTTA TAGGTT 3323 3480 HBV  [4] TGATGATGGTGCGCAGACCATCATC CATCAT 3496 4415 HBV  [4] TATAGAGAGAGAGAGAAGA CTCTAT TCTCTA 35865271 K6a [11] TTGATCCGGAGGTAGGTCTTT GGATCA CGGATC 3593  859 PLK1 [12]TTGGTATTCAGTGTGATGA ATACCA AATACC 3636 3304 APOB [13]TTACTCTCAAACTTTCCTC AGAGTA GAGAGT 3768 3885 XIAP  [9]TATTGTAATGGGCTCTGTC TACAAT TTACAA 4118 5055 E6/E7 [14]TGCCTTGGCAAACTTTCTT CAAGGC CCAAGG 4247 5408 EGFR1 [15]ACCAATTTATGCCTACAGC AATTGG AAATTG 4273 6322 HBV  [4]TTTGCTCTGTAGCAGCAGCTT GAGCAA AGAGCA 4298 5604 HTT  [1]CCAATCTCAAAGTCATCAA AGATTG GAGATT 4391 4652 AuRkb [15]TAGTTATTCAGGAAGTCTA ATAACT AATAAC 4421 4198 APOB [13]AATCAAGTAGATCCTCCTCC CTTGAT ACTTGA 4458 5308 AuRkb [15]TGCATCTCCTTGTCTACGC AGATGC GAGATG 4488 5464 bcl-xl  [2]TCAAGCTCTGCAAACCAGA AGCTTG GAGCTT 4547 4427 CCR5 [10]ATGATGATGGTGCGCAGAC TCATCA ATCATC 4561 3496 HBV  [4]TCTTCTAGCGTTGAAGTACTG TAGAAG CTAGAA 4583 4684 HTT  [1]TCTTCTAGCGTTGAATTACTG TAGAAG CTAGAA 4583 4684 HTT  [1]GAATTGTTGCTGGTTGCACTC ACAATT AACAAT 4647 4904 EGFR1 [15]TAGGACTAGTCACTTGTGC AGTCCT TAGTCC 4652 2822 K6a [11] TATAATGCTCAGCCTCAGACATTAT GCATTA 4672 3567 K6a [11] TTTGATTTGTAGCAGCAGCTT AATCAA AAATCA4735 6429 HTT  [1] TTTTATCTGTAGCAGCAGCTT GATAAA AGATAA 4785 4877 HTT [1] GAGTCTCTTGTTCCGAAGC GAGACT AGAGAC 4790 5151 VEGF [16]TATCACTCTATTCTGTCTC AGTGAT GAGTGA 4846 4396 Survivin  [9]TCACCTTCAAACTATGTCC AAGGTG GAAGGT 4852 4063 XIAP  [9]ATTGTCTTCAGGTCTTCAGTT AGACAA AAGACA 4855 5748 KSP [12]GCACTCCAGGGCTTCATCG GGAGTG TGGAGT 4944 5515 VEGF [16]AAGCCCCGAAAACCGGCTT GGGGCT CGGGGC 5090 2013 c-myc  [7]TTGTCCAGGAAGTCCTCAAGTCT TGGACA CTGGAC 5201 4750 PKN3 [17]CCAAGGCTCTAGGTGGTCA GCCTTG AGCCTT 5235 5726 bcl-xl  [2]GCACCACTAGTTGGTTGTC GTGGTG AGTGGT 5363 4425 TNFa [18]TCATCTCAGCCACTCTGCTTT GAGATG TGAGAT 5464 5351 DYT1 [19]GTCATCTCAGCCACTCTGCTT AGATGA GAGATG 5535 5464 DYT1 [19]AATGCAGTATACTTCCTGA CTGCAT ACTGCA 5549 6053 HIV [10]CACAATGGCACAGACTTCCAA CATTGT CCATTG 5565 4226 HTT  [1]CACAATGGCGCAGACTTCCAA CATTGT CCATTG 5565 4226 HTT  [1]TCTCCTCAGCCACTCTGCTTT GAGGAG TGAGGA 5692 5714 DYT1 [19]CTCCTCAGCCACTCTGCTTTT TGAGGA CTGAGG 5714 6646 DYT1 [19]TTCCTCAAATTCTTTCTTC TGAGGA TTGAGG 5714 5047 Survivin  [9]TTGTACATCATAGGACTAG TGTACA ATGTAC 5725 4158 K6a [11] TTGTCTTTGAGATCCATGCAAGACA AAAGAC 5748 5347 TNFa [18] TCAGCCCACACACAGTGCTTTG GGGCTG TGGGCT5938 5481 ID2 [20] TAACAAGCCAGAGTTGGTC CTTGTT GCTTGT 6008 4183 MAP4K4[18] TTCCAGAATTGATACTGACTT TCTGGA TTCTGG 6027 6482 CCR5 [21]TTTCCCTTGGCCACTTCTG AGGGAA AAGGGA 6352 5684 MAP4K4 [18]AAGCAGAGTTCAAAAGCCCTT TCTGCT CTCTGC 6576 6743 bcr-abl [22]TTGGGGATAGGCTGTCGCC TCCCCA ATCCCC 6591 3615 HCV [23]ATCTTCAATAGACACATCGGC TGAAGA TTGAAG 6618 5729 SOD1 [24]TTCCCCAGCTCTCCCAGGC TGGGGA CTGGGG 6649 6671 CCR5 [10]TTCCCCAAACCTGAAGCTC TGGGGA TTGGGG 6649 6070 HIV [10] TTCTTCTCATTTCGACACCAGAAGA GAGAAG 6650 6048 CCR5 [10] GTCCTGGATGATGATGTTC CCAGGA TCCAGG 68195883 VEGF [16] ATTTCAGGAATTGTTAAAG CTGAAA CCTGAA 6935 5757 APOB [13]CTTTCAGACTGGACCTCTC CTGAAA TCTGAA 6935 6689 Livin  [9]ACTGAGGAGTCTCTTGATCTT CCTCAG TCCTCA 6986 5833 CD4 [21]AAGCAAAACAGGTCTAGAATT TTTGCT TTTTGC 7110 6603 PCSK9  [3]CCCTCCCTCCGTTCTTTTT GGGAGG AGGGAG 7153 6058 c-myc  [7]GTTGTTTGCAGCTCTGTGC AAACAA CAAACA 7213 5301 E6/E7 [14]ATTCTCTCTGACTCCTCTC AGAGAA GAGAGA 7338 5454 CCR5 [10]TAATACAAAGACCTTTAAC TGTATT TTGTAT 7651 6954 HBV  [4]TATTTAAGGAGGGTGATCTTT TTAAAT CTTAAA 7880 6154 PLK1 [12]AAGAAATCATGAACACCGC ATTTCT GATTTC 8000 4935 ID2 [20] TAAACAAAGGACGTCCCGCTTGTTT TTTGTT 8980 8926 HBV  [4] AATTTTTCAAAGTTCCAAT AAAAAT GAAAAA 96788159 APOB [13]

REFERENCES CITED IN TABLE 6

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Example 2 Therapeutic siRNAs

Using the method described in Example 1 above, additional “safe seed”sequences were determined for the target genes indicated in Table 7below.

TABLE 7 Target ID NO. Gene Human Target site SEQ ID NO HDS1 HTTGTCGTGGCTCGCATGGTCGAT SEQ ID NO: 34 HDS2 HTT ATCCCGGTCATCAGCGACTATSEQ ID NO: 35 HDS3 HTT CTGCTTCTTTGTCAGCGCGTC SEQ ID NO: 36 HDS4 HTTGCGGGGCAGCAGGAGCGGTAG SEQ ID NO: 37 HDS5 HTT TTCCTCTTGTTTACGACGTGASEQ ID NO: 38 HDS6 HTT TGGGATGTAGAGAGGCGTTAG SEQ ID NO: 39 HDS7 HTTTCCCTTGGAATGCATATCGCT SEQ ID NO: 40 HDS8 HTT AACGTGGACCTGCCTACGGAGSEQ ID NO: 41 HDS9 HTT AGGGACAGTACTTCAACGCTA SEQ ID NO: 42 HDS10 HTTTGGGGACAGTACTTCAACGCT SEQ ID NO: 43 HDS11 HTT AAGGAGTTCATCTACCGCATCSEQ ID NO: 44 HDS12 HTT GAGCTGGCTCACCTGGTTCGG SEQ ID NO: 45 HDS13 HTTCTGCCCCAGTTTCTAGACGAC SEQ ID NO: 46 HDS14 HTT TGCCCCAGTTTCTAGACGACTSEQ ID NO: 47 HDS15 HTT GCCCCAGTTTCTAGACGACTT SEQ ID NO: 48 HDS16 HTTCCCCAGTTTCTAGACGACTTC SEQ ID NO: 49 HDS17 HTT CAGCTACCAAGAAAGACCGTGSEQ ID NO: 50 HDS18 HTT CTGCTGTGCAGTGATGACGCA SEQ ID NO: 51 HDS19 HTTATGGAGACCCACAGGTTCGAG SEQ ID NO: 52 HDS20 HTT TTCCGTGTGCTGGCTCGCATGSEQ ID NO: 53 HD521 HTT TCCGTGTGCTGGCTCGCATGG SEQ ID NO: 54 HDS22 HTTCTGGCTCGCATGGTCGACATC SEQ ID NO: 55 HDS23 HTT CACCCTTCAGAAGACGAGATCSEQ ID NO: 56 HDS24 HTT AACCTTTTCTGCCTGGTCGCC SEQ ID NO: 57 HDS25 HTTGAGGATGACTCTGAATCGAGA SEQ ID NO: 58 HDS26 HTT CCGGACAAAGACTGGTACGTTSEQ ID NO: 59 SCA1.S1 ATXN1 AAGCAACGACCTGAAGATCGA SEQ ID NO: 60 SCA1.S2ATXN1 CTGGAGAAGTCAGAAGACGAA SEQ ID NO: 61 SCA1.S3 ATXN1AACCAAGAGCGGAGCAACGAA SEQ ID NO: 62 SCA7.S1 ATXN7 ACGGGACAGAATTGGACGAAASEQ ID NO: 63 SCA7.S2 ATXN7 GTGGAAAAGATTCATCCGAAA SEQ ID NO: 64 SCA7.S3ATXN7 CAGGGTAGAAGAAAACGATTT SEQ ID NO: 65 SCA7.S4 ATXN7CGGCTCAGGAAAGAAACGCAA SEQ ID NO: 66 SCA2.S1 ATXN2 CCCCACATGGCCCACGTACCTSEQ ID NO: 67 SCA2.S2 ATXN2 ATCCAACTGCCCATGCGCCAA SEQ ID NO: 68 SCA2.S3ATXN2 CGCCAATGATGCTAATGACGA SEQ ID NO: 69 SCA2.S4 ATXN2CAGCCCATTCCAGTCTCGACA SEQ ID NO: 70 SCA2.S5 ATXN2 ACCCCACATGGCCCACGTACCSEQ ID NO: 71 SCA2.S6 ATXN2 AGCCCATTCCAGTCTCGACAA SEQ ID NO: 72 SCA2.S7ATXN2 TCCCAATGATATGTTTCGATA SEQ ID NO: 73 SCA2.S8 ATXN2TCCCAATGATATGTTTCGATA SEQ ID NO: 74

Example 3 Preclinical Safety of RNAi-Mediated HTT Suppression in theRhesus Macaque as a Potential Therapy for Huntington's

To date, a therapy for Huntington's disease (HD), a genetic,neurodegenerative disorder, remains elusive. HD is characterized by cellloss in the basal ganglia, with particular damage to the putamen, anarea of the brain responsible for initiating and refining motormovements. Consequently, patients exhibit a hyperkinetic movementdisorder. RNA interference (RNAi) offers therapeutic potential for thisdisorder by reducing the expression of HTT, the disease-causing gene. Wehave previously demonstrated that partial suppression of both wild-typeand mutant HTT in the striatum prevents behavioral and neuropathologicalabnormalities in rodent models of HD. However, given the role of HTT invarious cellular processes, it remains unknown whether a partialsuppression of both alleles will be safe in mammals whoseneurophysiology, basal ganglia anatomy, and behavioral repertoire moreclosely resembles that of a human. Here, we investigate whether apartial reduction of HTT in the normal non-human primate putamen issafe. We demonstrate that a 45% reduction of rhesus HTT expression inthe mid- and caudal putamen does not induce motor deficits, neuronaldegeneration, astrogliosis, or an immune response. Together, these datasuggest that partial suppression of wild-type HTT expression is welltolerated in the primate putamen and further supports RNAi as a therapyfor HD.

Huntington's disease (HD) is a fatal, dominantly inherited,neurodegenerative disorder caused by an expanded trinucleotide (CAG)mutation in the HTT gene on chromosome 4. The encoded protein, mutanthuntingtin (mHTT), contains an expanded polyglutamine stretch at theN-terminus, conferring a toxic gain of function. Over time, mHTT inducesthe formation of inclusions, cellular dysfunction, and neurodegenerationthroughout the basal ganglia and overlaying cortex. Cell loss in HD isaccompanied with upregulation of reactive astrocytes (astrogliosis) andactivation of microglia, the resident immune cells of the brain.Although cell loss is observed in multiple brain regions, neuropathologyis most pronounced in the medium-sized spiny neurons of the putamen andthe caudate, regions of the brain which are critical for the initiationand refinement of motor programs, procedural learning, and variousaspects of cognitive function. Accordingly, HD patients are afflictedwith involuntary hyperkinetic movements of the torso, arms, legs, andface (known as chorea) with concomitant gait and coordinationdifficulties, working memory deficits, and a variety of emotionaldisturbances.

To date, HD remains incurable. While several therapies have shownpromise in rodent models of the disease, including glutamateantagonists, bioenergetic supplements, caspase inhibitors,antihistaminergic agents (HORIZON trial) and fetal tissuetransplantation, none have made a significant impact on diseaseprevention or extension of life span when evaluated in clinical trials.As a result, current treatment strategies are primarily aimed atpalliative care to treat disease symptoms and improve end-stage qualityof life measures. With the elucidation of the causative HD mutation in1993, therapies can now be tailored toward reducing expression of thedeleterious gene itself, which may have a higher clinical impactcompared to strategies aimed at targeting downstream consequences ofmHTT.

Recently, it has become clear that endogenous, small microRNAs (miRNAs)play a vital role in regulating the expression of genes duringdevelopment, throughout adulthood and can contribute to disease states.Endogenous miRNA machinery can be co-opted and used to suppress genes ofinterest. Exogenous expression of engineered miRNAs as triggers for RNAinterference (RNAi) confers a robust decrease in gene expression and hasbeen investigated as a therapeutic tool to silence expression of diseasealleles. Inarguably, the preferred mechanism to treat HD would be tospecifically target the mutant allele while leaving the normal alleleintact. As a proof-of-principle, the benefit of allele-specificsilencing has been demonstrated by our laboratory members and others inrodent models of HD, wherein inhibitory RNAs were designed to silencethe human mHTT transgene and not endogenous mouse Htt. (Huang, et al.(2007). High-capacity adenoviral vector-mediated reduction of huntingtinaggregate load in vitro and in vivo. Hum Gene Ther 18: 303-311; Franichet al. (2008). AAV vector-mediated RNAi of mutant huntingtin expressionis neuroprotective in a novel genetic rat model of Huntington's disease.Mol Ther 16: 947-956; Harper et al. (2005). RNA interference improvesmotor and neuropathological abnormalities in a Huntington's diseasemouse model. Proc Natl Acad Sci USA 102: 5820-5825) Additionally,several single nucleotide polymorphisms (SNPs) that differentiate up to80% of diseased and normal alleles have been identified in the humanpopulation. (Pfister, et al. (2009). Five siRNAs targeting three SNPsmay provide therapy for three-quarters of Huntington's disease patients.Curr Biol 19: 774-778) However, the utility of these SNPs for RNAi-basedsilencing strategies have not been tested in vivo and importantly, willbe unusable for a significant number of HD patients.

Thus, an alternative strategy is to partially reduce expression of boththe mutant and normal allele in regions of the brain most affected bythe disease, a therapy that would be applicable to all HD patients.Because normal HTT has been found to play a functional role in the adultbrain, with proposed roles in mediating transcription and axonaltransport, nonallele-specific RNAi treatment for HD must demonstratetherapeutic benefit of reducing the mutant allele, as well as the safetyand tolerability of partially suppressing the normal allele. Over thepast half-decade, we have used recombinant adeno-associated viralvectors (rAAV) to deliver RNAi silencing constructs to the striatum andshowed that a 60% reduction of human mHTT and endogenous wild-type mouseHtt was well tolerated and prevented motor and neuropathologicaldeficits in transgenic mouse models of HD. Additionally, lentiviraldelivery of inhibitory RNAs in a rat model of HD conferred a 35%knockdown of Htt gene expression (both mutant and wild-type alleles) andwas safe and beneficial (both neuroanatomical and behavioral benefits)out to 9 months after injection. Furthermore, heterozygous Htt knockoutmice are phenotypically normal, and humans with only one copy of HTT(50% reduction of normal HTT production) show no abnormal behavioraldeficits, suggesting that nonallele-specific reduction of HTT expressionmay be safe.

While findings from rodent models are encouraging, it is essential toevaluate the safety of partial HTT suppression in an animal that moreclosely resembles humans with regards to the size, anatomy, andneurophysiology of its basal ganglia as well as its behavioralcapabilities prior to RNAi evaluation in human HD patients. Therefore,in this study, we assessed the safety of reduced HTT expression in therhesus macaque putamen. We demonstrate a partial, sustained HTTreduction in the putamen without the development of abnormal motorphenotypes, altered circadian behavior, fine motor skill deficits,neuronal loss, gliosis, or an immune response, thus bringing RNAi closerto the clinic as a potential therapy for HD.

Results

AAV2/1 Distribution and HTT Suppression in the Putamen

A sequence that silences mouse, rhesus, and human HTT and a controlsequence were cloned into an artificial miRNA backbone based on miR-30and subsequently cloned into AAV, serotype 1, vectors. (Boudreau, R L,Monteys, A M and Davidson, B L (2008). Minimizing variables amonghairpin-based RNAi vectors reveals the potency of shRNAs. RNA 14:1834-1844) Expression of the HD-specific miRNA (miHDS1) and the controlmiRNA (miCONT) was driven by a mouse U6 promoter. Enhanced GFP (eGFP)was driven from a cytomegalovirus (CMV) promoter to allow for assessmentof vector distribution following injection into the putamen. Both miHDS1(targeting a sequence in exon 52 of rhesus HTT mRNA) and miCONT (acontrol miRNA) were designed using “safe seed” guidelines to optimizesafety and minimize potential off-target gene silencing.

Prior to in vivo assessment in the rhesus macaque putamen, we firstverified HTT mRNA suppression by in vitro transfection of AAV shuttleplasmids expressing miHDS I, miCONT, or eGFP in human HEK293 cells aswell as rhesus primary fibroblasts generated at the Oregon NationalPrimate Research Center (50% and 32% reduction of relative HTT/18S mRNAexpression, respectively). Additionally, 60% silencing of striatal HttmRNA expression, without toxicity, was verified 4 weeks followinginjection of AAV2/1-miHDS1 injections into both wild-type and BACHDtransgenic mice.

Following verification of effective HTT mRNA suppression in vitro and inmice, eleven rhesus macaques received bilateral, MRI-guided stereotaxicinjections of either AAV2/1-miHDS1eGFP (therapeutic miRNA, n=4),AAV2/1-miCONT-eGFP (control miRNA, n=4) or AAV-eGFP (viral vectorcontrol, n=3) into the commissural and postcommissural putamen(posterior half of the entire putamen). Animals were assessed prior toand for six weeks postsurgery on a variety of general behavior and motorskill assays and euthanized for molecular (tissue punches taken from theleft hemisphere) and histological analyses (immuno-stained sectionsthrough the right hemisphere). Putamen samples transduced with AAV2/1(2×4 mm) were obtained from the left hemisphere of unfixed, coronalbrain slabs at necropsy. Quantitative polymerase chain reaction (QPCR)using primers flanking the miHDS1 targeting site in exon 52 demonstrateda significant reduction of rhesus HTT mRNA transcripts (45%, P<0.01)following injection with AAV1-miHDS1 compared to AAV-eGFPcontrol-treated putamen. We have previously demonstrated, in separateexperiments, that similar levels of silencing of either mutant human orwild-type Htt transcripts in mouse striatum cause marked reductions inthe respective proteins. (McBride et al. (2008). Artificial miRNAsmitigate shRNA-mediated toxicity in the brain: implications for thetherapeutic development of RNAi. Proc Natl Acad Sci USA 105: 5868-5873;Boudreau et al. (2009). Nonallele-specific silencing of mutant andwild-type huntingtin demonstrates therapeutic efficacy in Huntington'sdisease mice. Mol Ther 17: 1053-1063) eGFP immunohistochemistry wasconducted to assess viral vector distribution throughout the basalganglia. eGFP-positive cells were observed throughout the mid- andposterior putamen, indicating accurate needle placements during surgery.Immunofluorescence staining, using eGFP fluorescence as a reference,demonstrated AAV2/1 transduction in dopamine- and cAMP-regulatedneuronal phosphoprotein (DARPP-32)-positive medium spiny projectionneurons, choline acetyltransferase (ChAT)-positive large, cholinergicinterneurons, and glial fibrillary acid protein (GFAP)-positiveastrocytes throughout the putamen. eGFP-positive cells did notco-localize with IBA-1-stained microglia. In addition to eGFP-positiveneurons, astrocytes and fibers observed in the putamen, eGFP-positivecell bodies, and fibers were also seen in other regions of the basalganglia which receive projections from and project to the putamen. Theseinclude the internal and external segments of the globus pallidus), thesubthalamic nucleus (fibers only), and the substantia nigra parsreticulata. eGFP expression in the cortex was limited to the needletracts, suggesting that AAV2/1 was not transported anterogradily andretrogradily to the cortex, as was observed in other regions.

Unbiased stereology was employed to quantify the area fraction ofputamen containing eGFP-positive cells and fibers using serial sectionsstained with anti-eGFP antibody. Results demonstrated an area fractionof eGFP-positive cells in the commissural and postcommissural putamen of30±2.0% for AAV-eGFPinjected animals, 29±3.0% for AAV-miCONT-injectedanimals, and 30±3.0% for AAV-miHDS1 animals with no significantdifferences between groups (P>0.05). Additionally, quantification of theestimated volume of putamen containing eGFP-positive cells and fiberswas performed. The mean estimated volume of transduced putamen was1.0e11±1.7e10 μm³ for AAVGFP-injected animals, 8.5e10±5.6e9 μm³ forAAV-miCONT injected animals, and 9.9e10±1.6e10 μm³ for AAV-miHDS1animals. No significant difference in volume was found between treatmentgroups (P>0.05). A three-dimensional model of AAV2/1-transduced putamen(right hemisphere only) was created for each animal using StereoInvestigator software. The 3D rendering allows for the visualization ofthe three injection sites as well as the spread of vector followingsurgery. The anterior-posterior (A-P) distribution of eGFP-positivecells, a one-dimensional measure of AAV2/1 distribution from rostral tocaudal, was determined from one hemisphere of each of the elevenAAV2/1-injected animals. The mean A-P distribution for transducedputamen was 10.0±1.0 mm for AAV-GFP-injected animals, 9.5e10±1.0 mm forAAV-miCONT animals, and 9.5±0.58 mm for AAV-miHDS1 animals with nosignificant differences in spread between groups (Table 8, P>0.05).

TABLE 8 Measurement of anterior-posterior spread of eGFP-positiveregions of the putamen in individual animals injected with AAV2/1-eGFP(n = 3), AAV-miHds1 (n = 4), or AAV2/1-micont (n = 4) Animal Id Group APspread (mm) Rh24522 AAV2/1-eGFP 10.0 Rh24906 AAV2/1-eGFP 9.0 Rh25433AAV2/1-eGFP 11.0 Mean ± SD  10 ± 1.0 Rh24277 AAV2/1-miHDS1 10.0 Rh24353AAV2/1-miHDS1 9.0 Rh24530 AAV2/1-miHDS1 9.0 Rh25300 AAV2/1-miHDS1 10.0Mean ± SD  9.5 ± 0.58 Rh24377 AAV2/1-miCONT 9.0 Rh25150 AAV2/1-miCONT11.0 Rh25388 AAV2/1-miCONT 9.0 Rh25416 AAV2/1-miCONT 9.0 Mean ± SD 9.5 ±1.0

HTT Suppression does not Induce Motor Skill Deficits

To assess whether partial HTT suppression in the putamen, a region ofthe brain heavily involved in initiating, executing, and refining motormovement, induces motor perturbations, a variety of behavioral assayswere used to evaluate the monkeys prior to and for six weeks followingsurgery. We chose behavioral assays that allowed for the detection ofchanges in whole body movements in the homecage over 24-hour spans, morespecific coordinated movements of the arms and legs and learned tasksrequiring higher levels of dexterity of the forearms and digits.

To collect daytime and nighttime homecage activity, animals were fittedwith nylon or aluminum collars that housed an enclosed Acticalaccelerometer. All monkeys wore activity collars for 3 weeks prior tosurgery. The Actical monitor contains an omnidirectional sensor thatintegrates the speed and distance of acceleration and produces anelectrical current that varies in magnitude depending on a change inacceleration. The monitors were programmed to store the total number ofactivity counts during each 1-minute epoch. For daytime activity, arepeated measures ANOVA failed to detect significant differences betweentreatment groups, F (2,64)=0.17, P=0.84, suggesting that a partialreduction of HIT in the commissural and postcommissural putamen does notalter general homecage activity levels compared to controls. Asignificant effect was indicated for time, F (8, 64)=2.4, P<0.05, andHolms-Sidak pairwise comparisons showed that daytime activity during theweek immediately following surgery (+1) was significantly less than theactivity exhibited during week −2 (P<0.001) or week +5 (P<0.001), likelyowing to a small decrease in overall daytime activity while animalsrecovered from surgery. No group differences were observed (P=0.45).Likewise, for night time homecage activity, a repeated measures ANOVAindicated no significant differences between treatment groups F(2,64)=0.189, P=0.83, nor over time F (8,64)=1.43, P=0.20. Similarly, nointeraction was indicated (P=0.64). In addition to overall circadianhomecage activity, body weight from each animal was recorded at surgeryand at necropsy, and no decrease in weight was detected in any animal(P>0.05).

Potential changes in fine motor skills of left and right forelimbs anddigits were assessed using the Lifesaver test of manual dexterityoriginally described by Bachevalier et al. (1991, Agen monkeys exhibitbehavioral deficits indicative of widespread cerebral dysfunction.Neurobiol Aging 12: 99-111) and further modified by Gash and colleagues(1999, An automated movement assessment panel for upper limb motorfunctions in rhesus monkeys and humans. J. Neurosci Methods, 89:111-117). Animals were transported from their homecage to a WisconsinGeneral Testing Apparatus in a separate behavioral room and trained toremove hard, round treats from a straight medal rod (straight post). Forthe straight post, animals were assessed 2 weeks prior to surgery tocollect baseline data and weekly for 6 weeks after surgery (two trialsper forelimb each day, twice a week). No statistical difference wasdetected in the latencies to remove stimuli from posts between the rightand left hands. Consequently, the right and left hand data werecollapsed, and averages were used for all analyses. A repeated measures,two-way ANOVA indicated a significant main effect of time over thetesting trials, F (6, 48)=27.5, P<0.0001, indicating that animals fromall treatment groups removed the treat from the post with shorterlatencies (faster performance) as the study progressed. By contrast, nosignificant effects were found between the treatment groups, F(2,48)=0.07, P=0.99, nor for an interaction (P=0.55), indicating thatAAV-miHDS1-treated animals performed with the same speeds as animalsfrom both control groups. For the Lifesaver task using the question markshaped post, animals received no training prior to surgery so that wecould assess each animal's ability to learn a new and more difficulttask (procedural learning) following AAV-miHDS1 injection into theputamen. Beginning 2 weeks following surgery and each week thereafter,latencies to successfully remove each treat off the question mark shapedpost were recorded (two trials per forelimb each day, twice a week). Arepeated measures two-way ANOVA failed to indicate significantdifferences between groups, F (2,28)=0.573, P=0.58, or over testingtrials, F (4,28)=0.61, P=0.652 nor for an interaction (P=0.93). Thesedata show that animals from all treatment groups were able to completethe question mark post task with equal speed and that HTT suppressiondid not alter the ability of the AAV-HDS1-treated animals to (1) learn anew behavioral task or (2) exhibit fine motor skills on a difficult taskcompared to controls.

Additionally, we developed a non-human primate-specific, preclinicalmotor rating scale (MRS) that was modified from the Unified Huntington'sDisease Rating Scale used for evaluating motor performance in HDpatients. We designed the MRS to specifically assess putamen-basedbehavioral phenotypes in monkeys including horizontal and verticalocular pursuit, treat retrieval with both forelimbs, ability to bearweight on both hindlimbs, posture, balance, and startle response. Inaddition, the scale includes negative motor phenotypes seen in HD orcases of putamen dysfunction including bradykinesia (slowness ofmovement), dystonia (involuntary, sustained muscle contraction), andchorea (involuntary, hyperkinetic movement) of each limb and trunk.Possible scores ranged from 0 (normal phenotype) to 3 (severely abnormalphenotype) for a total of 72 possible points. Animals were rated bythree, independent observers blind to treatment group and familiar withnonhuman primate behavioral repertoires; inter-rater reliability was100%. All animals were evaluated in their homecage and were rated onceprior to surgery and each week thereafter for the duration of the study.Kruskal-Wallis statistical analysis revealed a significant differencebetween the three treatment groups (H(2)=9.30, P=0.010). However, thisdifference is due to one AAVmiCONT-injected animal that exhibited a verymild but progressive dystonia in one hind leg (animal 25150). A Dunn'spairwise comparison shows no difference between AAV-miHDS1-injectedanimals compared to AAV-eGFP-injected controls, demonstrating that apartial HTT suppression in the mid- and posterior putamen did not alternormal putamen-based behavior nor induce diseased phenotypes commonlyseen with neuronal dysfunction or degeneration in the putamen.

HTT Suppression does not Cause Neuronal Degeneration, Gliosis, orInflammation

To address whether HTT reduction in cells of the putamen caused neuronaldegeneration, we evaluated potential neurotoxicity byimmunohistochemical staining for eGFP to identify transduced regions ofthe putamen, NeuN (neuronal marker), GFAP (astrocytic marker), and Iba1(microglial marker). Coronal brain section were stained using standardDAB immunohistochemistry, and adjacent sections were compared for signsof neuron loss, increases in astrocyte proliferation (reactiveastrocyosis) or increases in reactive microglia in AAV-miHDS1-treatedmonkeys compared to controls. Compared to AAV-eGFP- andAAV-miCONT-injected controls, AAV-miHDS1-injected animals showed no lossof NeuN-positive neurons in the putamen. Cresyl violet (Nissl) stainingof adjacent coronal brain section s further supported a lack of neuronalloss. To assess whether partial HTT suppression was associated withcellular dysfunction, in contrast to frank neuronal loss, we performedQPCR analysis for DARPP-32, a highly expressed protein in GABA-ergicprojection neurons of the putamen. DARPP-32 is a key mediator innumerous signal transduction cascades, and its downregulation has beenreported in cases of medium spiny neuronal dysfunction in the absence ofNeuN downregulation. Consequently, DARPP-32 is a valid and reliablereadout of neuronal function in the putamen. QPCR analysis of transducedregions of the putamen found no significant decrease of DARPP-32 mRNAexpression in monkeys injected with AAV-miHDS1 compared to controls(P>0.05).

Coronal stained sections from all treatment groups showed a mildincrease in GFAP-positive astrocytes in transduced regions, likely dueto the injection itself and not a reduction in HTT since equalastrocytosis was observed in all groups. IBA-1-stained sections fromanimals in each group showed no increases in activated microglia, exceptfor within the injection tracts, likely due to physical perturbation ofparenchyma by the needle. To further assess inflammation, expression ofthe pro-inflammatory cytokines interleukin 1-β (IL1-β) and tumornecrosis factor-α (TNF-α) was measured from transduced regions. Both ofthese cytokines are upregulated and released from astrocytes andmicroglia in response to distressed, neighboring neurons in the brain.QPCR analysis showed no significant increases in IL1-β (P>0.05) or TNF-α(P>0.05) in AAV-miHDS1-treated monkeys compared to AAVeGFP controlanimals. Interestingly, monkeys injected with AAV-miCONT showed asignificant decrease in TNF-alpha expression compared to bothAAV-eGFP-(P<0.05) and AAV-miHDS1-(P<0.05) animals.

Lack of Peripheral Immune Response Following AAV1-miRNA Delivery to thePutamen

Previous studies have shown that peripheral T cells infiltrate the brainfollowing injury or infection. Thus, in addition to assaying for localinflammatory and immune responses in the putamen, cell-mediated andhumoral responses were evaluated to determine whether AAV-mediatedsuppression of HTT induced peripheral immune responses. Relative CD4 andCD8 mRNA expression levels were determined by QPCR to address whetherAAV suppression of HTT induced infiltration of peripheral helper orcytotoxic T cells, respectively. No significant differences were seenbetween groups in either CD4 or CD8 mRNA expression in transducedputamen samples (P>0.05). Also, no inflammatory infiltrates were notedon Nissl-stained sections from treated animals. To test if anti-AAVantibodies were induced after injection, an in vitro neutralizingantibody (Nab) assay was performed on serum collected from each animalimmediately prior to surgery and at necropsy (6 weeks after injection).HuH7 cells were infected with AAV2/1 expressing LacZ in the presence ofserial dilutions of rhesus serum. The transduction assay showed that thecohort of rhesus macaques used for this study displayed varying levelsof neutralizing antibodies to AAV2/1 in their serum prior to surgeryranging from undetectable titers (<1:5) to the highest titer of 1:160.Four of the 11 animals showed increases in AAV2/1 Nab levels at necropsybut these increases were minor (two- to fourfold). Neither presurgicalNab levels nor the fold change in Nab expression from presurgery tonecropsy correlated with levels of eGFP expression in the putamen(Pearson's correlation, r=−0.24, P=0.49 and Spearman correlation(r=0.01, P=0.9, respectively).

DISCUSSION

Here, we present novel data showing that a partial reduction of HTTexpression in the rhesus macaque putamen is well tolerated out to 6weeks after injection. We used a multifaceted approach to assess theability of RNAi to reduce HTT and address whether such suppression wouldinduce behavioral or neuropathological consequences by combining assaysof gross and fine motor skills with postmortem immunohistochemical,stereological, and molecular analyses of neuronal, glial, and immuneprofiles. Our silencing construct, miHDS1, was designed such that thetarget mRNA sequence displays homology to rodent, rhesus macaque, andhuman HTT. Therefore, HTT reduction and tolerability can be seamlesslyevaluated in transgenic mice and non-human primates. Importantly, thesame sequence evaluated preclinically may be utilized to evaluate safetyof HTT suppression in a phase 1 clinical trial.

The selection of our injection sites in the mid- and posterior putamenwas based upon the primate putamen's functional rostral-caudal gradient.Lesions of the posterior aspect of the putamen with excitotoxins orlentiviral-mediated delivery of mutated Htt elicit hyperactivity,choreiform movements, stereotypes, and/or dyskinetic movements of thelimbs (either spontaneously or following apomorphine administration).Correspondingly, we have previously observed motor dysfunction detectedvia the Lifesaver assay and MRS following moderate neuronal loss in themid- and posterior putamen (unpublished results from our laboratory). Bycontrast, lesions of the anterior putamen fail to produce similardyskinesias. These disparate effects correspond with the inputs to themid- and posterior putamen from the primary sensorimotor corticesincluding the premotor and supplementary motor areas as well as theprimary motor area. By contrast, the anterior primate putamen receivescortical inputs from the frontal association areas, the dorsolateralprefrontal cortices, and the pre-supplementary motor area. Consequently,to assess the tolerability of partial HTT suppression in the mid- andposterior putamen, we employed three behavior tests that assessputamen-associated behaviors. First, to assess potential changes ingeneral activity, we continually assessed homecage activity over theduration of the experiment using omnidirectional activity monitorsplaced in collars on the animals. No differences in daytime or nighttimeactivity were found between groups.

In an effort to detect more subtle abnormalities of limb use, muscletone, eye movements, posture or balance, we devised a MRS based upon theclinical Unified Huntington Disease Motor Rating Scale. Our rubricassessed 24 discrete behaviors and revealed that 10 of the 11 animalsshowed no behavior anomalies. One AAV-miCONT-injected control animal(no. 25150) displayed a mild dystonia in his left leg. The increasedmuscle tone in the leg was noted on day 12 subsequent to surgery and maybe the result of trauma, infection from the surgical procedure or aperturbation in the putamen due to the injection itself.

To challenge the functional integrity of the mid- and posterior putamenand its circuits, all animals were trained to perform the Lifesavertask. The task requires the animals to rapidly perform a sequence ofmuscle movements in the arm, hand, and fingers to obtain a reinforcer.For the straight post task, animals were trained for 21 days prior tothe initiation of the experiment in an effort to increase animals'efficiency, skill, and speed of performance. Evidence suggests thatover-learned sequential hand movements require the functional integrityof the posterior sensorimotor putamen in monkeys and in humans.Consistent with homecage activity and motor ratings, there were nodifferences in the performance of the straight post task between theHDS1 animals and the controls, again supporting the notion thatknockdown of normal HTT in the mid- and posterior putamen does notsignificantly diminish the functional integrity of its circuits.

In contrast to the posterior regions, the anterior and mid-levels of theputamen are known to play an essential role in learning new handmovement sequences. Whereas our intraputamen injections did not coverthe entire anterior putamen, eGFP transfection was observed in sections˜3 mm rostral to the anterior commissure. Thus, to assess the potentialdisruption of a procedural learning circuit, we presented a novelquestion mark-shaped post 2 weeks following surgery. Despite never beingtrained on the distinctively shaped post, all groups successfullylearned to perform the task at equal rates, suggesting that the relevantputamen circuits were functionally intact. Thus, consistent withhomecage activity and motor rating data, partial knockdown of endogenousHTT in the mid- and posterior putamen did not diminish the execution ofa previously learned motor task nor impair the acquisition of novelmanual dexterity task.

We observed robust eGFP expression in both neurons and astrocytesthroughout the commissural and postcommissural putamen followinginjection of each construct. Here, AAV2/1 transduced bothDARPP-32-positive medium spiny projection neurons and ChAT-positivelarge, aspiny interneurons. While medium spiny neurons show the mostdramatic cell loss in HD, the large cholinergic neurons are alsoaffected by mHTT. Cholinergic interneurons exhibit decreased levels ofChAT and decreased levels of acetylcholine release in transgenic mousemodels of HD as well as HD patients. In contrast to the findingspresented here, and by other groups (Dodiya, et al. (2010). Differentialtransduction following basal ganglia administration of distinctpseudotyped AAV capsid serotypes in nonhuman primates. Mol Ther 18:579-587) using eGFP as a reporter gene, primarily astrocytictransduction was seen following injection of AAV2/1 expressing humanizedrenilla GFP (hrGFP) into the cynomolgus macaque putamen. (Hadaczek, etal. (2009). Transduction of nonhuman primate brain with adeno-associatedvirus serotype 1: vector trafficking and immune response. Hum Gene Ther20: 225-237.) Additionally, a robust anti-hrGFP antibody response wasalso observed, along with CD4⁺ lymphocyte infiltration and localmicroglial responses, suggesting that hrGFP may be less well toleratedin the non-human primate putamen compared to eGFP.

Our finding that AAV2/1 transduces astrocytes, as well as neurons, inthe putamen may provide additional benefit in animal models of thedisease and in HD patients. While most therapeutic strategies for HDhave targeted vulnerable neurons, a growing body of evidence hasdemonstrated that astrocytes also contain mHTT-positive inclusionbodies. Astrocytes expressing mHTT contain fewer glutamate transportersand are less capable of protecting against glutamate-mediatedexcitotoxicity. Additionally, Bradford and colleagues demonstrated thatdouble transgenic HD mouse models expressing truncated mHTT in bothneurons and glia exhibit more severe neurological symptoms than miceexpressing mHtt in neurons alone (Bradford, et al. (2010). Mutanthuntingtin in glial cells exacerbates neurological symptoms ofHuntington disease mice. J Biol Chem 285: 10653-10661). Thus, partiallysuppressing HTT in both neurons and glia may have a more robust clinicalimpact.

eGFP-positive neurons and fibers, but not glia, were also found in theinternal and external globus pallidus as well as the substantia nigrapars reticulata, indicating retrograde and anterograde transport of thevector, respectively. eGFP-positive fibers only were seen in thesubthalamic nucleus. These findings may have important clinicalimplications for HD as these regions of the basal ganglia also undergomHTT-induced cell loss and gliosis. Injections into a single brainregion (putamen) may have the capability of therapeutically targetingmultiple vulnerable brain regions. Specifically, transduced neurons inthe globus pallidus and substantia nigra should also expressHTT-specific miRNAs and may therefore be amenable to RNAi therapy.Ongoing analyses in our laboratory are currently investigating thelevels of miRNA expression and concomitant levels of HTT mRNAsuppression in these brain regions.

Our immunohistochemical and molecular results demonstrate a significant45% decrease in HTT, a level of suppression which has shown therapeuticbenefit in mouse models of HD without inducing toxicity (targeting bothmutant and wild-type alleles). This level of suppression did not induceNeuN-positive cell loss or downregulate DARPP-32 expression. We detecteda very mild upregulation of GFAP-positive astrocytes in transducedregions of the putamen. Because astrogliosis was detected in animalsfrom all three groups, it was not due to a reduction in Htt expressionin neighboring neurons. Rather, the mild astrogliosis was likely due tothe injection itself. Because brains were evaluated at 6 weeks afterinjection, this low level of gliosis would be predicted to decrease overtime. Importantly, we saw no upregulation in reactive microglia orpro-inflammatory cytokine expression which would be predicted toincrease if HTT reduction induced neural toxicity.

Recombinant AAV gene transfer to the intact CNS has been shown to elicita minimal T cell-mediated response without a salient plasmacell-mediated immune response in preclinical animal studies.Additionally, encouraging findings from recent early-stage gene therapyclinical trials for Canavan's Disease (CD), Parkinson's Disease (PD),and Leber's congenital amaurosis (LCA) wherein AAV, serotype 2, wasdirectly injected into the brain parenchyma (CD, PD) or the retina(LCA), demonstrated only mild increases in Nab levels after injectionwith no signs of inflammation or adverse neurological events. Theresults here further support these findings and demonstrate thatalthough monkeys had a range of preexisting, circulating Nab levelsprior to surgery (from undetectable up to 1:160), there was no majorincrease in Nab levels (two- to fourfold maximum) 6 weeks afterinjection. Moreover, despite the minor increase in Nab levels in 4/11animals, there was no correlation of Nab levels with the area fractionof GFP⁺ cells in the putamen. Interestingly, the presence of preexistingNab titers at the upper range of what we report has been shown tosubstantially abrogate gene expression following systemic, intravascularinjection of varying serotypes of AAV to target either brain orperipheral tissues. Our data are encouraging and suggest that eventhough NHPs and humans have natural circulating antibodies to AAV2/1, aswell as other serotypes, a preexisting antibody load, at least up to thevalues reported here, will not limit gene transfer and should not be anexclusion criteria for clinical trials involving direct braininjections.

In summary, our results in the rhesus macaque brain further support andextend previous experiments in rodents demonstrating the safety andefficacy of a nonallele-specific HTT reduction. These findings, alongwith the well-established safety profile of rAAV in early phase clinicaltrials for a variety of neurological disorders, underscore the potentialof viral-mediated RNAi as a therapy for HD.

Materials and Methods

Animals.

Eleven normal adult rhesus macaques of Indian origin (male, 7-10 kg)were utilized in this study. All monkeys were maintained one per cage ona 12-hour on/12-hour off lighting schedule with ad libitum access tofood and water. All experimental procedures were performed according toOregon National Primate Research Center and Oregon Health and ScienceUniversity Institutional Animal Care and Use Committee and InstitutionalBiosafety Committee approved protocols.

RNAi Constructs and Viral Vector Production.

All siRNAs were generated using an algorithm developed to reduce theoff-targeting potential of the antisense sequences. (See Example 1above) siRNA sequences targeting either a sequence in exon 52 of mouse,rhesus, and human huntingtin or a control siRNA were embedded into anartificial miRNA scaffold comparable to human miR-30 to generate miHDS1(pri: 5′-AGUGAGCGAUGCUGGCUCGCAUGGUCGAUACUGUAAAGCCACAGAUGGGUGUCGACCAUGCGAGCCAGCACCGCCUACU-3′, predicted antisense sequence in bold,nucleotides 5-83 of SEQ ID NO: 33) or miCONT (pri: 5′-AGUGAGCGCAGCGAACGACUUACGCGUUUACUGUAAAGCCACAGAUGGGUAAACGCGUAAGUCGUUCG CUACGCCUACU (SEQ IDNO: 200), predicted antisense sequence in bold). Artificial miRNA stemloops were cloned into a mouse U6 expression vector, and the expressioncassettes were subsequently cloned into pFBGR-derived plasmids whichcoexpress CMV-driven GFP. Shuttle plasmids (pAAVmiHDSI-GFP andpAAVmiCONT-GFP) contain the respective transcriptional units which areflanked at each end by AAV serotype 2 145-bp inverted terminal repeatsequences. rAAV production was performed using the Baculovirus AAVSystem. (Smith, R H, Levy, J R and Kotin, R M (2009). A simplifiedbaculovirus-AAV expression vector system coupled with one-step affinitypurification yields high-titer rAAV stocks from insect cells. Mol Ther17: 1888-1896.) Sf9 insect cells were infected with a baculovirusexpressing AAV rep2, AAV cap 1, and adenovirus helper proteins and asecond baculovirus expressing the miRNA and eGFP flanked by the AAV2ITR's. The cell lysate was run through an iodixanol gradient (15%-60%wt/vol), and the iodixanol fraction containing the rAAV particles wasfurther purified using a Mustang-Q ion exchange filter membrane. rAAVparticle titer was determined by QPCR and FACS analysis. Vectors weregenerated by the Gene Transfer Vector Core at the University of Iowa andsent to the Oregon National Primate Research Center for injections.Twelve hours before surgery, all viral vector preps were dialyzedagainst Formulation Buffer 18 (Hyclone) to remove salts (3 total hoursof dialysis) and diluted to a final titer of 1e12 vg/ml.

Magnetic Resonance Imaging and Stereotaxic Surgery.

Immediately prior to surgery, animals were anesthetized with KetamineHCL (10 mg/kg), transported to the MRI, intubated and maintained on 1%isoflurane vaporized in oxygen for the duration of the scan. Animalswere placed into an MRI-compatible, stereotaxic surgical frame; aT1-weighted magnetic resonance image (MRI) was conducted to obtainsurgical coordinates (Siemens 3.0 T Trio MR unit). After scanning,animals were taken directly into the operating room and prepped forsterile surgery. Each animal received three microinjections perhemisphere (six injections total): the first 1 mm rostral to theanterior commissure (12 μl) and the two remaining injections (12 μl and10 μl, respectively) spaced 3 and 6 mm caudal to the first injection.Animals were injected with 1e12 vg/ml of either AAV2/1-miHDS1-eGFP(n=4), AAV2/1-miCONT-eGFP (n=4) or AAV2/1-eGFP (n=4) at a rate of 1μl/minute, and the needle was left in place for an additional 5 minutesto allow the injectate to diffuse from the needle tip. Aftermicroinjections were completed, the skull opening was filled withgelfoam and the incision closed.

Behavioral Analysis

General homecage activity: All animals were fitted with either nylon oraluminum collars (Primate Products) with Actical accelerometers(Respironics) mounted onto the frame. Each Actical monitor contained anomnidirectional sensor that integrated the speed and distance of wholebody acceleration and produced an electrical current that varies inmagnitude depending on a change in acceleration. The monitor wasprogrammed to store the total number of activity counts for each1-minute epoch. Animals wore activity collars 24 hours a day, 7 days aweek for 3 weeks prior to surgery and each week thereafter for theduration of the study.

MRS: Three independent observes, blinded to group identity, assessedhomecage behavior weekly. Twenty-four separate putamen-associatedbehaviors were rated including horizontal and vertical ocular pursuit,treat retrieval with both forelimbs, ability to bear weight on bothhindlimbs, posture, balance, startle response and bradykinesias,dystonias and choreas of each limb and trunk. A score of 0 indicated anormal phenotype while a score of 3 indicated severely abnormalphenotypic movements. All animals were evaluated on the MRS prior tosurgery to obtain baseline scores and once per week for the duration ofthe study.

Lifesaver test: Animals were trained to thread edible, hard treats froma straight metal rod (straight post) and then tested on their ability toremove treats from the straight post and a question mark-shaped post.All manual dexterity tasks were presented in a Wisconsin general testingapparatus (WGTA) and the latency to successfully retrieve the treat wasmeasured separately for the left and right forelimbs. Animals weretrained for 21 days on the straight post. Then, 2 weeks of baseline datawere collected on the straight post only. Two weeks following surgery,animals were tested twice per week on both the straight post and thequestion mark-shaped post. On testing days, each animal was placed intothe WGTA and their movements recorded on digital video. Each hand wastested two times with a time limit of 5 seconds for the straight postand 10 seconds for the question mark post to complete the task. Thelatency to remove each treat was assessed via Sony PMB software withmillisecond measuring capability at a later time.

Necropsy and Tissue Processing.

Six weeks after surgery, animals were sedated with Ketamine and thendeeply anesthetized with sodium pentobarbital followed byexsanguination. Brains were perfused through the ascending carotidartery with 2 l of 0.9% saline, removed from the skull, placed into anice-cold, steel brain matrix and blocked into 4-mm-thick slabs in thecoronal plane. Tissue punches used for molecular analyses were obtainedfrom each animal's left hemisphere of the transduced putamen (slabs wereplaced under the fluorescent scope to verify eGFP-fluorescing regions)and immediately frozen in liquid nitrogen to preserve DNA, RNA, andprotein. Slabs were subsequently postfixed in 4% paraformaldehyde forhistological analyses.

Quantitative Real-Time PCR.

RNA was isolated from tissue punches taken from eGFP-positive putamenusing the Qiagen RNeasy kit, as per the manufacturer's instructions, andreverse transcribed with random primers and Multiscribe reversetranscriptase (Applied Biosystems, Carlsbad, Calif.). Relative geneexpression was assessed via QPCR by using TaqMan primer/probe sets forDARPP-32 (Hs00259967_m1), CD4 (Rh02621720_m1), CD8 (Rh02839719_m1),IL1-β(Rh02621711_m1), or TNF-α(Rh02789784). All values were quantifiedby using the ΔΔCT method (normalizing to 18S) and calibrated toAAV-GFP-injected putamen. Primers for rhesus HTT mRNA quantificationwere designed to flank the miHDS1 binding site in Exon 52 using PrimerExpress (Applied Biosystems): Forward: 5′-CGGGAGCT GTGCTCACGT-3′ (SEQ IDNO: 201), Reverse: 5′-CATTTCTACC CGGCGACAAG-3′ (SEQ ID NO:202)), andexpression was assessed using SYBR Green detection. At the conclusion,dissociation curve (melting curve) analysis was performed to confirmspecific amplification.

Immunohistochemical Analyses.

40-μm-thick, free-floating coronal brain sections were processed forimmunohistochemical visualization of eGFP expression (eGFP, 1:000,Invitrogen), neurons (NeuN, 1:1000, Millipore), reactive astrocytes(GFAP, 1:2000, DAKO), or microglia (Iba1, 1:1,000; WAKO) by using thebiotin-labeled antibody procedure. Following endogenous peroxidaseinhibition and washes, tissues were blocked for 1 hour in 5% donkeyserum, and primary antibody incubations were carried out for 24 hours atroom temperature. Sections were incubated in donkey anti-rabbit oranti-mouse biotinylated IgG secondary antibodies (1:200; VectorLaboratories, Burlingame, Calif.) for 1 h at room temperature. In allstaining procedures, deletion of the primary antibody served as acontrol. Sections were mounted onto gelatin-coated slides andcoverslipped with Cytoseal 60 (Thermo Scientific, Waltham, Mass.).Images were captured by using an Olympus BX51 light microscope and DP72digital color camera, along with an Olympus DP Controller software.

Immunofluorescence Analyses.

40-μm-thick, free-floating coronal brain sections were processed forimmunofluorescent visualization of medium spiny projection neurons(DARPP-32, 1:25, Cell Signaling, Danvers, Mass.), large cholinergicneurons (ChAT, 1:500, Millipore, Billerica, Mass.), reactive astrocytes(GFAP, 1:1000, DAKO, Carpinteria, Calif.), or microglia (Iba1, 1:500;WAKO, Richmond, Va.). Following washes, tissues were blocked for 1 hourin 5% donkey serum, and primary antibody incubations were carried outfor 24 hours at room temperature. Sections were incubated in donkeyanti-rabbit or anti-goat Alexa-546 conjugated secondary antibodies(1:500; Invitrogen, Carlsbad, Calif.) for 1 hour at room temperature.Sections were mounted onto gelatin-coated slides and coverslipped withSlowfade Gold anti-fade mounting media containing DAPI (Invitrogen).Images were captured at ×20 magnification using a Leica SP5 confocalmicroscope.

Stereological Determination of Vector Distribution.

The Area Fraction Fractionator (Microbrightfield) was used to quantifythe fraction of eGFPpositive cells in the putamen (right hemisphereonly). Every 12th coronal section (1/2 series, 40-μm-thick sections)through the putamen containing GFP⁺ cells was selected for analysis. Theputamen was outlined under ×2 magnification, and a rectangular latticeof points was overlaid. One marker was used to select points that fellwithin the region of interest (putamen), and a second marker was used toselect points that fell within the subregion of interest (containedGFP-positive cells). The counting frame area was 1000×1000 μm, XYplacement was 1600×1600 μm, and grid spacing was 120 μm. The areafraction estimation of GFP⁺ cells in the putamen was determined bydividing the area of GFP⁺ cells by the area of the putamen and estimatesprovided were averaged from all sections quantified. A 3D reconstructionof the eGFP-transduced putamen was created using StereoInvestigatorsoftware by aligning contours from each section from the rostral tocaudal putamen and placing skins over each. The anterior to posteriorspread of eGFP transduction was determined by locating the most rostraland caudal sections through the putamen containing GFP and using acombined MRI and histology atlas of the rhesus monkey brain (Saleem andLogothetis) to identify the distance between the two (1 mm resolution).

Neutralizing Antibody Assay.

Whole blood was collected in red top Vacutainer Serum Tubes (BD) fromanimals prior to surgery and at necropsy, serum was collected followingcentrifugation at 2500 rpm for 20 minutes and stored at −80° C. untilanalysis. Serum was sent to the Immunology Core at the University ofPennsylvania for analyzing AAV2/1 antibody levels via an in vitrotransduction assay. A 96 well plate was seeded with Huh7 cells andinfected with AAV2/1-LacZ and serial dilutions of pre- and postsurgeryrhesus serum. Values reported are the serum dilution at which relativeluminescence units (RLUs) were reduced by 50% compared to virus controlwells (no serum sample). The lower limit of detection was a 1/5dilution, and anti-AAV2/1 rabbit serum was used at a positive control.

Statistical Analysis.

All statistical analyses were performed by using SigmaStat statisticalsoftware (SYSTAT). QPCR analyses for HTT, DARPP-32, CD4, CD8, IL1-β, andTNF-α expression, as well as Area Fraction Fractionator analyses, wereperformed by using a one-way ANOVA. Upon a significant effect,Bonferroni post hoc analyses were performed to assess for significantdifferences between individual groups. For homecage activity andLifesaver test analyses, a two-way, repeated measures ANOVA using groupand time as variables was run to determine differences between groups orover time. Post hoc analyses were performed when statisticallysignificant differences were detected. For MRS analyses, aKruskal-Wallis test was run followed by a Dunn's pairwise comparison todetect differences between groups. Correlational data between the areafraction of GFP in the putamen and presurgical Nab levels weredetermined using a Pearson's correlation for parametric data.Correlational data between the area fraction of GFP in the putamen andthe fold change of Nab titers pre- and postsurgery were determined usinga Spearman correlation for nonparametric data. In all cases, P<0.05 wasconsidered significant.

All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain preferred embodiments thereof,and many details have been set forth for purposes of illustration, itwill be apparent to those skilled in the art that the invention issusceptible to additional embodiments and that certain of the detailsdescribed herein may be varied considerably without departing from thebasic principles of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1-72. (canceled)
 73. A nucleic acid encoding an artificial primary miRNAtranscript (pri-miRNA) comprising, in order of position, a 5′-flankingregion, wherein the 5′-flanking region comprises a 5′-bulge sequencepositioned upstream from a 5′-joining sequence; a non-guide region,wherein the 5′-joining sequence is contiguously linked to the non-guideregion; a loop region; a guide region; and a 3′-flanking region, whereinthe guide region comprises a sequence at least 80% identical tocgaccaugcgagccagca (miHDS.1 guide. SEQ ID NO:7) and the non-guide regionis at least 80% complementary to the guide region.
 74. The nucleic acidof claim 73, wherein the guide region consists of 18-30 nucleotides. 75.The nucleic acid of claim 73, wherein the 5′ joining sequence consistsof 5-8 nucleotides.
 76. The nucleic acid of claim 73, wherein the5′-bulge sequence consists of 1-10 nucleotides.
 77. The nucleic acid ofclaim 73, wherein the 5′-flanking region further comprises a 5′-spacersequence positioned upstream from the 5′-bulge sequence.
 78. The nucleicacid of claim 77, wherein the 5′-spacer sequence consists of 10-12nucleotides.
 79. The nucleic acid of claim 77, further comprising a5′-upstream sequence positioned upstream from the 5′-spacer sequence.80. The nucleic acid of claim 79, wherein the 5′-upstream sequenceconsists of 30-2000 nucleotides.
 81. The nucleic acid of claim 73,wherein the 3′-flanking region comprises a 3′-joining sequencecontiguously linked to the guide region.
 82. The nucleic acid of claim81, wherein the 3′-joining sequence consists of 5-8 nucleotides.
 83. Thenucleic acid of claim 81, wherein the 3′-joining sequence is at leastabout 85% complementary to the 5′-joining sequence.
 84. The nucleic acidof claim 81, further comprising a 3′-bulge sequence positioneddownstream from the 3′-joining sequence.
 85. The nucleic acid of claim84, wherein the 3′-bulge sequence consists of 1-10 nucleotides.
 86. Thenucleic acid of claim 84, further comprising a 3′-spacer sequencepositioned downstream from the 3′-bulge sequence.
 87. The nucleic acidof claim 86, wherein the 3′-spacer sequence consists of 10-12nucleotides.
 88. The nucleic acid of claim 86, further comprising a3′-downstream sequence positioned downstream from the 3′-spacersequence.
 89. The nucleic acid of claim 88, wherein the 3′-downstreamsequence is about 30-2000 nucleotides in length.
 90. The nucleic acid ofclaim 73, wherein the loop region is from 15-25 nucleotides in length.91. An expression cassette comprising a promoter contiguously linked tothe nucleic acid of claim
 73. 92. A vector comprising the expressioncassette of claim
 91. 93. The vector of claim 90, wherein the vector isan adeno-associated virus (AAV) vector.
 94. The vector of claim 93,wherein the AAV is AAV1, AAV2, AAV4, AAV5, or AAV2/1.
 95. An isolatedmicroRNA molecule comprising the nucleic acid of claim
 73. 96. A methodof inducing RNA interference comprising administering to a subject aneffective amount of the nucleic acid of claim
 73. 97. A method oftreating a subject with Huntington's Disease, comprising administeringto the subject the nucleic acid of claim 73 so as to treat theHuntington's Disease.